**2. Materials and Methods**

#### *2.1. Experimental Site and Design*

Healthy seeds of 22 green super rice (GSR) varieties and four local controls (IR-6, Kisan Basmati, Kashmir Basmati, and NIAB-B-2016) were obtained from CAAS, China, and NIGAB, Pakistan, respectively. Previously, Kashmir Basmati was reported as a heattolerant genotype, while the NIAB-B-2016 as a susceptible variant [23]. All four controls are normally high-yielding rice varieties grown in Pakistan. The seeds were sown at National Institute for Genomics and Advanced Biotechnology (NIGAB), National Agricultural Research Center (33.684◦ N and 73.048◦ E), Islamabad, Pakistan. During the rice growing season of 2020, the seeds of selected lines were placed in trays containing 128 wells filled with a mixture of soil, sand, and peat moss, all containing essential nutrients. The transplantation of the 35-days-old seedlings was carried out on 20 July 2020. For this purpose, randomized complete block design (split plot) with three replicates was followed. Two sets of 26 genotypes were transplanted in the field (one for control and one for heat). Each plot consisted of five rows with 10 plants each. Row-to-row and plant-to-plant distance were kept at 30 cm [4]. Recommended agronomic practices were followed.

At the flowering stage (pre-anthesis), a tunnel was prepared to cover the plants with a polythene sheet and high temperature was maintained (40–45 ◦C) to apply heat stress from 10:00 a.m. to 03:00 p.m. After 03:00 pm, polythene sheets were removed daily to reduce the temperature (25–35 ◦C). Morpho-physiological parameters were recorded at maturity from the central five randomly-selected plants in order to remove the border effect [47]. After heat exposure, a pollen fertility test was performed for all genotypes to screen out heat-susceptible genotypes. All fresh leaf samples were collected and stored at −80 ◦C for further analysis.
