The study material in the GWAS panel with 282 advanced breeding lines of bread wheat genotypes from IARI stress breeding program was selected to map the genomic regions responsible for grain iron and Zinc content under drought and heat stress treatments.

Phenotypic data:

The GWAS panel was evaluated at IARI, New Delhi - DL (28.6550° N, 77.1888° E, MSL 228.61 m) under Irrigated (IR), Restricted Irrigated (RI) and Late sown (LS) treatment conditions with augmented RCBD design. Data was collected on Grain Iron and Grain zinc content along with thousand-grain weight. Around 20 g of grain sample from each of 282 genotypes from the GWAS panel under all three conditions were used for phenotyping GFeC and GZnC through high-throughput Energy Dispersive X-ray Fluorescence (ED-XRF) machine (model X-Supreme 8000; Oxford Instruments plc, Abingdon, United Kingdom) calibrated with glass beads-based values. To record TGW, manual counting of grains was followed and the weight of the grains was recorded in grams with an electronic balance.

Genotypic data:

Genomic DNA of the GWAS panel was extracted from the leaves of seedlings by Cetyl Trimethyl Ammonium Bromide (CTAB) method. The panel was genotyped using Axiom Wheat Breeder’s Genotyping Array (Affymetrix, Santa Clara, CA, United States) having 35,143 genome-wide SNPs. The monomorphic, markers with minor allele frequency (MAF) of <5%, missing data of >20%, and heterozygote frequency >25% were removed from the analysis. The remaining set of 10546 high-quality SNPs was used in GWAS analysis.

All the materials were evaluated at IARI New Delhi (Indian Agricultural Research Institute, research farm located 28°38′30.5″N, 77°09′58.2″E, 228 m AMSL) with three conditions viz., Timely sown irrigated (IR), Timely sown Drought (RI) and Late sown condition (LS). A total of 6 irrigations was given for the irrigated and late sown trials. Whereas one irrigation before and one after the sowing was provided for restricted irrigated trials. IR and RI trials were sown on the first fortnight of November whereas LS trials was sown in second fortnight of December to expose plants to the natural heat in later growing periods. Experiment was conducted in Augmented RCBD design with four checks and six blocks with plot size of 1 m2 each.

Phenotyping

Twenty random spikes from each line were collected in clean polyethylene bags and hand threshed. Grain Fe and Zn contents (in mg/Kg) were estimated with 20 gram seeds of each line through high-throughput Energy Dispersive X-ray Fluorescence (ED-XRF) machine (model X-Supreme 8000; Oxford Instruments plc, Abingdon, United Kingdom) calibrated with glass beads based values (Paltridge et al., 2012). Thousand kernel weight was measured by manual counting. The GWAS panel under all the three conditions were phenotyped for GFeC, GZnC and TGW. Phenotypic data were analysed using the r package ‘augmentedRCBD’ (Aravind et al. 2021) for ANOVA and adjusted means for each genotype under study. The z test was used to compare the statistically significant differences between control and treatment means.