The functional rice variety ZBXN 1 was bred by the Special Rice Research Institute of Yushan County, Jiangxi Province and selected from the parents of the August glutinous rice and the mutated purple glutinous rice. The process of selection and breeding was as follows: the local variety of August glutinous rice was crossed with the local variety of purple glutinous rice as the parent, and the F₁ generation was backcrossed with the parent to obtain BC₁F₁. In 2006, the strain was approved by the Jiangxi Provincial Variety Approval Committee and named Ganwannuo6.

The RILs used in this study consisted of 81 lines, which were obtained by crossing ZBXN 1 as the female plant and MH63 as the male plant to obtain the F₁, and then backcrossing the F₁ with MH63 to obtain BC₁F₁, and thereafter using the single seed descent method to obtain. In this experiment, three generations of BC₁F₆, BC₁F₇ and BC₁F₈ of RILs were finally selected as the study materials, noted as RIL6, RIL7 and RIL8, and all seeds were collected for the determination of anthocyanin and total flavonoid contents in brown rice.

The experiment referred to the “Determination of anthocyanins in foods of plant origin by high performance liquid chromatography” (NY/T 2640-2014), and the content of anthocyanins in brown rice was determined by high performance liquid chromatography method. Accurately weighed 2g brown rice sample into 50ml colorimetric tube, added the prepared anthocyanin extraction solution and fixed the volume to the scale. Vortex shook for 1min and then used ultrasonic extraction for 1h. After extraction, put the colorimetric tube into boiling water bath and hydrolyzed for 1h, took it out and left it cool down, and then added the extraction solution to the scale in constant volume again. Subsequently, let it stand for a few minutes, took the supernatant and passed it through 0.45um aqueous filter membrane into the machine directly. In the chromatographic conditions, the mobile phase was adjusted to 1% acetonitrile formate solution and the gradient elution conditions were adjusted from a complex elution gradient to a simple gradient, which saved time and effort in the whole process on the basis of ensuring the accuracy of experimental data.

In this experiment, referring to the industry standard “Determination of total flavonoids in buckwheat and its products” (NY/T 1295-2017), the pretreatment method was partially optimized, and the total flavonoid content in brown rice was determined by spectrophotometric method. Accurately weighed 2g brown rice sample and placed it in a 100 mL stoppered triangular flask, absorbed 30 mL of aqueous methanol solution, covered the stopper tightly, placed the triangular flask in a constant temperature water bath shaker at 65 °C and shook it at 160 r/min for 2 h. The filtrate was placed in a 50 mL volumetric flask. The filter paper and residue were washed with aqueous methanol solution. The filtrate was combined, cooled to room temperature, and then fixed the volume to the sacle with aqueous methanol solution. Meanwhile, a standard curve was made with 0.0500 mg·mL^-1 of Rutin working solution. Absorbed 1 mL of the solution to be measured into a 10 mL volumetric flask, added 2 mL of 0.1 mol·L^-1 Aluminium trichloride solution and 3 mL of 0.1 mol·L^-1 potassium acetate solution according to the preparation method of Rutin working solution, and fixed the volume to the scale with methanol aqueous solution. Then shook it well, and left it at room temperature for 30 min, and centrifuged it at 4000 r·min^-1 for 10 min. The absorbance value was measured the absorbance value A420 at 420 nm. The total flavonoid content was calculated by substituting it into the equation of standard curve.

To investigate the anthocyanin development rule of parents ZBXN 1 and MH63, the experiment observed the phenotypes of parents’ grains at different periods (8, 16, 24 and 32 days after flowering) and measured their anthocyanin content. The color of the grains of ZBXN 1 gradually deepened as developing, with the darkest color of the seeds at 24 days after flowering and eventually fading to purple, however, MH63 did not have the color change of ZBXN 1 and it eventually showed the color of conventional rice ( Figure 1 ).

The experiment further analyzed the anthocyanin content of ZBXN 1 at different filling stages by liquid chromatography, and the results showed an S-shaped curve variation ( Figure 2 ). After flowering, the content of anthocyanins, from 0 to 16 days, showed a stable change and low content, at 8 d there are only peony pigments, while from 16 to 24 days anthocyanins showed a significant increase in content and the content was four times higher than that before 16 days. The content decreased slightly from 24 to 32 days at maturity. The anthocyanin content was half of that of 24 days. In summary, it can be seen that the anthocyanin synthesis of ZBXN 1 was significant during the developmental period from 16 to 24 days during the filling period, and the anthocyanin content decreased slightly when it reached the maturity period.

The contents of ZBXN 1 and MH63 showed opposite S-shaped curve changes at different filling stages ( Figures 3A, B ), the total flavonoid content of ZBXN 1 decreased from 0.06% to 0.023% at 8-16 days after flowering, and increased sharply to a maximum of 0.229% from 16 to 24 days after flowering, but it decreased to 0.094% from 24 to 32 days after flowering. The total flavonoid content of MH63 increased from 0.095% to 0.192% from 8 to 16 days after flowering, and then decreased gradually to 0.115% from 16 to 32 days after flowering. Finally, the total flavonoid content at maturity was 0.082%.The total flavonoid contents of ZBXN 1 and MH63 were similar at the final maturity stage. Therefore, it can be seen that total flavonoids of ZBXN 1 and MH63 showed an inverse relationship from 0 to 24 days after flowering and were consistent from 24 to 32 days after flowering.

The trend graph of anthocyanin content results at different filling stages of ZBXN 1 was compared with its trend graph of total flavonoid content ( Figures 2 , 3A ). From the graph, we can see that anthocyanin and total flavonoid content showed mostly the same trend throughout the rice growth and development to maturity, and both showed an increasing trend at 8 d after the beginning of grouting, except that the total flavonoid content developed relatively faster than the anthocyanin. From 8 to 16 d, the total flavonoid was on a declining curve, but anthocyanin content tended to be stable. From 16 to 24 d, both contents showed sharp increases consistently, and from 24 to 32 d, they decreased to a stable state. This finding demonstrates that ZBXN 1 is a very high-quality rice variety that can be widely used as an intermediate material for breeding different functional special crops.

The RIL population was constructed by crossing ZBXN 1 as the female plant and MH63 as the male plant, and 81 long dominant materials were selected by measurement. In this study, the anthocyanin and total flavonoid contents of 81 RILs lines of different generations were determined by liquid chromatography.

Excluding the samples with 0 mg/kg of detection, the anthocyanin content of the 41 lines was finally determined and compared. The study of anthocyanin content in different generations of RIL populations showed that the overall analysis of anthocyanin genetic stability in different generations of the same RIL line was relatively stable ( Figure 4 ). Among Z1, Z2, Z3, Z4 and Z6 with anthocyanin content over 1500 mg/kg, the content of anthocyanin in Z4 showed the largest change, and the content of anthocyanin decreased step by step. Z1 showed the smallest change, indicating that Z1 had better stability and higher overall content than Z4, while Z2 and Z3 were relatively better. Among Z5, Z7, Z8, Z9 and Z10 with anthocyanin content ranging from 500 mg/kg to 1500 mg/kg, Z10 had the biggest change in anthocyanin content, and also showed a decreasing trend, while other samples were relatively stable. The relative content of anthocyanin in Z11 to Z41 with 0 mg/kg~500 mg/kg was stable, and the anthocyanin content in Z13 was basically unchanged in three different generations, showing a super stable inheritance. Therefore, the anthocyanin content of 41 RILs samples was relatively stable at different generations. In addition, the total content of anthocyanins in RILs population was less than 500 mg/kg, accounting for 75.61%. The content of 500 mg/kg-1500 mg/kg and higher than 1500 mg/kg accounted for about 12.2%. In conclusion, the anthocyanin content of RILs offspring is generally low, but the content of individual family samples is high and has a stable inheritance. The average anthocyanin content of parent ZBXN 1 was 319.31 mg/kg, and that of parent MH63 was 0 mg/kg. Z1 had the highest anthocyanin content, with a mean value of 1894.75 mg/kg, which was not only significantly better than that of the parents, but also had good stability.

It can be seen from the changes of total flavonoids content in different generations of each RILs sample that the overall total flavonoids are genetically stable ( Figure 5 ). Z25, where the total flavonoids content is more than 0.3% and the difference between the highest and lowest flavonoids content is 0.09%, which can be ignored, has good stability. Among Z11, Z24, Z39, Z41, Z49, Z52, Z61 with 0.2%-0.3% total flavonoids content, Z49 showed the largest content change, and the difference between the highest and lowest content was 0.15%, indicating poor genetic stability. The variation of total flavonoids content in Z39 and Z41 was 0.03%, which showed good stability. Samples with the content of 0 ~0.2% were stable. The average content of total flavonoids of parent ZBXN 1 was 0.15% and the content of parent MH63 was 0.13%. But the content of total flavonoids of the highest content of Z25 was 0.33%, which indicated that Z25 was obviously better than that of the parent, and has good stability.