**2.3. Estimation of grain protein content, iron and zinc concentrations**

Grain protein content was estimated with near infrared reflectance (NIR) spectroscopy of whole grains (ZX50 Portable Grain Analyzer, USA) using respective calibration software provided by Zeltex. Three repetitions were studied using 25 grains per line.

The measurements of grain iron and zinc concentrations were described in detail [33]. Briefly, grain samples (100-Gy- and 200-Gy-dosed M5 mutant lines and cv. Eritrosperumum-35) were washed with sodium dodecyl sulfate (0.1%) with the following several times washings in deionized water, dried, ground with a mixer mill (Retsch MM400 GmbH) and digested (0.2 g) with a mixture of nitric acid (65%, analytical grade) and hydrogen peroxide (30%) (5:1, v/v) using digestion automat K-438 and scrubber K-415 model triplescrub (BÜCHI Labortechnik AG CH-9230 Flawil 1/Switzerland). The sample was diluted to 20 mL with twice-distilled water. The Fe and Zn concentrations were analyzed using flame atomic absorption spectroscopy (Analytic Jena NovAA350, Germany). Estimation of mineral nutrients were checked against the certified reference values from the State standard samples LLC "HromLab," Zn 7837–2000, Fe 7835–2000 diluted by 0.3% HNO3. Three extracts and analysis repetitions were performed.
