*3.1. Assessment of Genetic Diversity Using Principal Component Analysis*

Principal component analysis (PCA) was performed to study the genetic differences among the genotypes, and trait–genotype biplots were constructed from data recorded under control and heat stress (Figure 1). Under control conditions, PC1 and PC2 captured the 31.7% and 26.8% of the total variations (Figure 1A). Our results explained that GY, HI, GW, and NDVI showed opposite response to GL, PH, TPP, and SY. The GSR lines were more conserved because they were clustered near the origin, while the check varieties Kisan Basmati, Kashmir Basmati, NIAB-B-2016, and IR-6 showed more genetic variability because they spread far away from the center of origin (Figure 1A). Similarly, under heat stress, PC1 and PC2 showed 34.8% and 21.7% variations, respectively (Figure 1B). PFP, NDVI, HI, and GY were in opposite direction to the rest of the studied parameters (TPP, PH, SY, GL, GW, and HSI). In contrast to control conditions, both the GSR and check varieties fall away from the origin, which suggests that genotypes were more responsive to heat stress compared to the control (Figure 1B). Importantly, NGSR-3, NGSR-13, Kashmir Basmati and IR-6 were found near the apex of the biplot under heat stress, suggesting that these genotypes have the distinct genetic potential of the best tolerance to heat stress compared to others (Figure 1B).

**Figure 1.** PCA under control (**A**) and heat stress (**B**) for 22 GSR lines and four controls and studied parameters. PH = Plant height (cm), TPP = Tillers per plant, GY = Grain yield per plant (g), SY = Straw yield per plant (g), HI = Harvest index, GW = 1000-Grain weight (g), GL = Grain length (mm), NDVI = Normalized difference vegetative index, PFP = Pollen fertility percentage, CMS = Cell membrane stability, and HIS = Heat susceptibility index.
