Mapping of the QTLs governing grain micronutrients and thousand kernel weight in wheat (Triticum aestivum L.) using high density SNP markers

The mapping population consists of 166 recombinant inbred lines (RILs) derived from a cross between HD3086 and HI1500.

Phenotypic data
The RILs population along with parents were evaluated under four conditions namely timely sown irrigation (TSIR) taken as control, timely sown restricted irrigation (TSRI), late sown irrigation (LSIR), and late sown restricted irrigation (LSRI) conditions at Delhi, and under restricted irrigation condition at Indore. From each plot, 20 random spikes were harvested and spikes from each plot were threshed separately. While cleaning, care was taken to prevent metal and dust contamination. The grain iron concentration (GFeC) and grain zinc concentration (GZnC) were measured using Energy Dispersive X-ray Fluorescence (ED-XRF) machine (model X-Supreme 8000 M/s Oxford Inc, USA).  The thousand kernel weight (TKW) was recorded by counting 1000 grains manually and weighted with an electronic balance.

Genotypic data
DNA was extracted from 21 days old seedlings using CTAB method (Murray and Thompson, 1980). Genomic DNA quality was determined using 0.8% agarose gel electrophoresis with λ DNA as the standard and quantified using nanodrop. The 35K SNP Axiom breeders’ array was used for genotyping of parents and the RILs population.

Methods
The RILs population along with parents were evaluated under four conditions namely timely sown irrigation (TSIR) taken as control, timely sown restricted irrigation (TSRI), late sown irrigation (LSIR), and late sown restricted irrigation (LSRI) conditions at Delhi, and under restricted irrigation condition at Indore. Two irrigations were given in TSRI, one at germination and other at 21 days after sowing; six irrigations were provided during cropping period in irrigated condition (TSIR). Late sown trials (LSIR and LSRI) were planted in second fortnight of December to expose plants to heat stress, and under LSRI condition irrigation was withheld to expose plants to both terminal heat and drought stress. The genotypes were evaluated in an alpha-lattice design with two replications. Each genotype was sown in 3 rows of 1 m each with 22.5 cm distance between rows and 10 cm distance between plants. Uniform agronomic practises were practiced for proper establishment of crop stand.

Phenotyping
From each plot, 20 random spikes were harvested and spikes from each plot were threshed separately. While cleaning, care was taken to prevent metal and dust contamination. The grain iron concentration (GFeC) and grain zinc concentration (GZnC) were measured using Energy Dispersive X-ray Fluorescence (ED-XRF) machine (model X-Supreme 8000 M/s Oxford Inc, USA).  The thousand kernel weight (TKW) was recorded by counting 1000 grains manually and weighted with an electronic balance. MetaRv6.0 (Multi Environment Trial Analysis with R) software (Alvarado et al., 2020) was used to calculate best linear unbiased predictors (BLUPs).