*2.1. Phenotypic Variation and Correlation Analysis*

In 2016 and 2017, the GPC of the HHZ/JZ1560 RIL population and the parents was measured by NIRS and KND, respectively. The descriptive statistics of phenotypic variation are shown in Table 1. Significant phenotypic differences were observed between the parents, and the GPC of JZ1560 was higher than that of HHZ in both years. Frequency distributions of GPC in the brown rice flour of RILs and the parents were plotted (Figure 1). The GPC of the RILs showed similar distribution either measured by the KND or NIRS method in each year. Phenotypic variation was continuously distributed with low skewness and kurtosis, showing a typical pattern of quantitative variation. Using the KND method, the GPC of the RILs showed a wide variation from 7.27% to 15.78% in 2016 and from 7.39% to 15.98% in 2017 (Figure 1). Continuous segregation and significant transgressive segregation at two directions suggested polygenic control underlying this trait.

**Table 1.** Descriptive statistics of the grain protein content (GPC, %) in the recombinant inbred line (RIL) population and the parents in the two years.


NIRS: near infrared reflectance spectroscopy; KND: Kjeldahl nitrogen determination; *p*: two-tailed *p* value of student's *t* test.

**Figure 1.** Frequency distributions of grain protein content (GPC) measured by near infrared reflectance spectroscopy (NIRS) and Kjeldahl nitrogen determination (KND) methods in the recombinant inbred line (RIL) population. Parental values are indicated by arrows.

Significant positive correlations between the GPC values measured by the two methods or in different years were found in the RIL population (Table 2). However, correlation coefficients for GPC determined by KND and NIRS methods as 0.966 in 2016 and 0.983 in 2017 were much higher than that of GPC measured with the same method in different years as 0.638 for NIRS and 0.631 for KND. This implied that GPC determined by NIRS was quite consistent with that by the KND method, and GPC was strongly influenced by environmental factors.

**Table 2.** Correlation coefficients of GPC between two methods and between different years from 280 RILs.


16NIRS, 17NIRS: GPC of 280 RILs in 2016 and 2017 measured by near infrared reflectance spectroscopy (NIRS); 16KND, 17KND: GPC of 280 RILs in 2016 and 2017 measured by Kjeldahl nitrogen determination (KND). \*\* highly significant correlations at the 0.01 level.
