*4.2. GPC Measurement*

GPC of brown rice flour for each RIL was carefully measured by the KND and NIRS methods, respectively. Rice grains were harvested after maturity and stored at room temperature for at least three months before the GPC measurement. The fully filled grains were de-hulled into brown rice by a rice huller (JLGJ-45, Taizhou, Zhejiang, China), then the brown rice was grinded into flour by a grinder (IKA TUBE-MILL, Staufen, Germany). Brown rice flour samples were directly used to determine GPC by NIRS (Foss, Sweden). Each sample was assayed twice and the mean values were used for further analysis. GPC was calculated according to the modified NIRS model constructed by Xie et al. [23].

For the KND method to measure GPC, 0.2 g brown rice flour, 1.0 g catalyst (CuSO4:Na2SO4 = 1:10) and 4.0 mL of H2SO4 were first added into a 100-mL digestion tube in turn. Then the mixture was immediately heated at 420 ◦C for 2 h in a digestion stove. After the solution being digested, the mixture was cooled to room temperature. Then, 10 mL ddH2O was added into the digestion tube. The mixed solution was analyzed using a Kjeltec 8400 Autoanalyzer (Foss, Sweden). GPC of the rice flour was calculated from the total N content by multiplying a conversion factor of 5.95 [30]. The assay for each sample was conducted with two replicates and the means were used for further analysis.
