
Rice used to be a major staple food crop of the world, especially in Asian countries including Korea. The history of rice introduction to Korean peninsula is estimated as the early Bronze age (Ahn 2010) resulting in diverse landraces in Korea (Lee
Rice grains, especially unpolished brown rice carrying nutrient-rich rice bran are well-known to contain various phytonutrients: ferulic acid, phenolics, anthocyanins, phytic acid, γ-oryzanol, octacosanol, dietary fiber, tocopherols, tocotrienols, and phytosterols, etc. (Zhou
Considering the long cultivation history of rice in Korea as a major food crop, the presence of diverse Korean landraces is a proof of continuous efforts and achievements in traditional rice breeding. Since ‘Namseon-1-ho’, the first modernized rice variety developed in 1932, more than 300 rice varieties for table and special purposes have ever been developed in Korea (Cho
A rice core collection consisting of 294 accessions were established by a Center of Crop Breeding on Omics and Artificial Intelligence, Kongju National University (Choi
Lipophilic phytonutrients such as tocopherols, tocotrienols, squalene, and phytosterols were quantified according to Mahmud
A one-step extraction/methylation protocol (Cho
Rice samples cultivated in 3 different locations were used as 3 biological replications for all phytonutrient analyses. Statistics software SPSS (version 25, IBM Corp., USA) were used to calculate basic statistics and correlation coefficients at
Among 8 vitamer forms of tocols, 4 major ones: α-tocopherol (T), γ-T, α-tocotrienol (T3), and γ-T3 were quantified for 157 Korean bred-rice varieties. The average contents of α-T, γ-T, α-T3, γ-T3, total T, total T3, and total tocols (vitamin E) were 11.9, 1.6, 10.0, 13.9, 13.4, 23.9, and 37.3 μg/g, respectively (Table 1), and α-T, α-T3 and γ-T3 consisted 32%, 27%, and 37% of total tocols. The range of minimum and maximum contents for α-T, α-T3, and γ-T3 were 3.0-21.6, 1.8-17.1, and 8.2-27.7 μg/g so that the range of total T and total T3, and total tocols were 6.4-22.9, 17.4-36.8, and 26.8-54.9 μg/g, respectively. The accessions which showed the highest and lowest content for each tocol are as followings; α-T (RWG225, 21.6 μg/g and RWG204, 3.0 μg/g), α-T3 (RWG287, 17.1 μg/g and RWG238, 1.8 μg/g), γ-T (RWG165, 8.2 μg/g and RWG153, 0.3 μg/g), and g -T3 (RWG163, 27.7 μg/g and RWG216, 8.2 μg/g) (Table 2). The tocols contents exhibited quite different compositional pattern between rice ecotypes. The average content of α-T in japonica-type varieties was 12.9 μg/g, which was about 2.5 times higher than that of indica-type ones. Similarly, α-T3 showed about 3 times higher contents in japonica-type rice (11.0 μg/g) compared to indica-type (3.8 μg/g), while γ-T and γ-T3 contents in japonica-type exhibited only 24% and 61% of indica-type varieties, respectively. These results are strongly related with ecotype-dependent differences in α-T/γ-T and α-T3/γ-T3 ratios in that japonica-type ones showed significantly higher α-T/γ-T (15.15) and α-T3/γ-T3 (0.95) ratio corresponding to 629% and 428% of indica-type ones, whose α-T/γ-T and α-T3/γ-T3 ratios were 2.41 and 0.22, respectively. These ecotype-dependent variations are responsible for bimodal distributions observed in histograms of α-T and α-T3 content as well as α-T/γ-T and α-T3/γ-T3 ratio (Fig. 1). Regardless of ecotypes, total tocopherol contents were always lower than total tocotrienol contents so that the T/T3 ratios in 137 japonica-, 21 indica-types, and all 157 accessions were 0.60, 0.40, and 0.57, respectively. Although total tocotrienol contents showed no difference between ecotypes, japonica-type varieties showed statistically higher total tocopherol (14.0 μg/g) and total tocols (37.8 μg/g) contents compared to indica-type accessions.
Table 1 . Statistics for tocopherol (T), tocotrienol (T3), squalene (SQ), campesterol (CA), sitosterol (SI), and stigmasterol (ST) contents (μg/g) in brown rice of 157 varieties bred in Korea.
Ecotype | Statistics | α-T | γ-T | α-T3 | γ-T3 | SQ | CA | SI | ST | αT/gT ratio | αT3/gT3 ratio | T/T3 ratio | Total T | Total T3 | Total tocols | Total phytosterols |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Indica | Case | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 |
Mean | 5.2 | 4.5 | 3.8 | 21.0 | 15.2 | 42.8 | 169.2 | 18.1 | 2.41 | 0.22 | 0.40 | 24.8 | 34.5 | 34.5 | 230.0 | |
SDz) | 2.5 | 1.9 | 2.6 | 4.2 | 14.3 | 9.3 | 18.2 | 2.4 | 4.40 | 0.24 | 0.11 | 3.7 | 4.5 | 4.5 | 23.2 | |
Median | 4.4 | 4.6 | 3.0 | 20.7 | 12.0 | 40.7 | 169.3 | 17.6 | 0.95 | 0.15 | 0.39 | 26.3 | 35.8 | 35.8 | 233.4 | |
Minimum | 3.0 | 0.8 | 1.8 | 13.4 | 8.1 | 27.8 | 141.1 | 14.6 | 0.59 | 0.10 | 0.24 | 19.3 | 26.8 | 26.8 | 192.6 | |
Maximum | 12.4 | 8.2 | 13.6 | 27.7 | 76.4 | 60.4 | 198.6 | 22.2 | 19.22 | 1.14 | 0.75 | 30.7 | 40.4 | 40.4 | 265.2 | |
RSDy) (%) | 49% | 41% | 70% | 20% | 94% | 22% | 11% | 13% | 183% | 107% | 28% | 15% | 13% | 13% | 10% | |
Japonica | Case | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 |
Mean | 12.9 | 1.1 | 11.0 | 12.8 | 38.3 | 42.0 | 162.6 | 20.3 | 15.15 | 0.95 | 0.60 | 23.7 | 37.8 | 37.8 | 224.9 | |
SD | 2.0 | 0.5 | 1.6 | 2.2 | 13.0 | 6.1 | 21.2 | 2.7 | 6.01 | 0.16 | 0.09 | 3.1 | 4.6 | 4.6 | 27.7 | |
Median | 12.8 | 1.0 | 11.0 | 12.5 | 36.2 | 41.6 | 159.7 | 20.2 | 14.30 | 0.94 | 0.59 | 23.3 | 37.9 | 37.9 | 224.4 | |
Minimum | 8.6 | 0.3 | 6.9 | 8.2 | 7.9 | 28.2 | 114.8 | 13.5 | 3.81 | 0.31 | 0.38 | 17.4 | 28.3 | 28.3 | 162.9 | |
Maximum | 21.6 | 2.9 | 17.1 | 22.5 | 78.4 | 71.4 | 235.3 | 27.7 | 46.57 | 1.56 | 0.95 | 36.8 | 54.9 | 54.9 | 320.2 | |
RSD (%) | 16% | 44% | 14% | 18% | 34% | 15% | 13% | 14% | 40% | 17% | 15% | 13% | 12% | 12% | 12% | |
Total | Mean | 11.9* | 1.6* | 10.0* | 13.9* | 35.2* | 42.1x) | 163.5x) | 20.0* | 13.45* | 0.85* | 0.57* | 23.8* | 37.3x) | 37.3* | 225.6x) |
SD | 3.4 | 1.4 | 3.0 | 3.8 | 15.3 | 6.6 | 20.9 | 2.8 | 7.26 | 0.30 | 0.12 | 3.2 | 4.7 | 4.7 | 27.1 | |
RSD (%) | 28% | 91% | 30% | 27% | 43% | 16% | 13% | 14% | 54% | 35% | 20% | 20% | 14% | 13% | 12% |
z)Standard deviation.
y)RSD: Relative standard deviation (%).
x)Not significant difference between ecotypes by t-test (
*Significant difference between ecotypes by t-test (
Table 2 . List of varieties which showed highest and lowest level of phytonutrients. The RWG accession numbers followed by its phytonutrient content are provided. Information on details of each RWG accession is provided in Supplementary Table S1.
Phytonutrient | Description | RWG accession number (phytonutrient content, in μg/g) | |||||||
---|---|---|---|---|---|---|---|---|---|
α-Tocopherol | Highest | 225 (21.6) | 179 (17.6) | 143 (16.8) | 198 (16.7) | 158 (16.6) | 291 (16.5) | 224 (16.5) | 221 (16.2) |
Lowest | 204 (3.0) | 205 (3.0) | 208 (3.2) | 209 (3.3) | 272 (3.4) | 210 (3.8) | |||
α-Tocotrienol | Highest | 287 (17.1) | 148 (14.3) | 155 (14.3) | 291 (14.0) | 150 (14.0) | |||
Lowest | 238 (1.8) | 165 (2.1) | 166 (2.1) | 208 (2.3) | 211 (2.5) | ||||
γ-Tocopherol | Highest | 165 (8.2) | 166 (8.0) | 206 (6.2) | |||||
Lowest | 153 (0.3) | 215 (0.4) | 240 (0.4) | 181 (0.4) | |||||
γ-Tocotrienol | Highest | 163 (27.7) | 276 (27.6) | 211 (25.2) | 292 (25.0) | 166 (24.7) | |||
Lowest | 216 (8.2) | 275 (8.8) | 280 (8.9) | 196 (9.2) | 264 (9.2) | 195 (9.4) | |||
Squalene | Highest | 225 (78.4) | 164 (76.4) | 222 (73.7) | 219 (72.5) | ||||
Lowest | 258 (7.9) | 209 (8.1) | 204 (8.6) | ||||||
Campesterol | Highest | 225 (71.4) | 292 (60.4) | 143 (57.8) | |||||
Lowest | 165 (27.8) | 268 (28.2) | 164 (28.5) | ||||||
Sitosterol | Highest | 187 (235.3) | 225 (224.8) | 290 (208.4) | 287 (208.4) | ||||
Lowest | 190 (114.8) | 185 (116.8) | 212 (121.4) | 199 (125.0) | 157 (126.9) | 193 (127.7) | |||
Stigmasterol | Highest | 293 (27.7) | 240 (26.7) | 139 (26.5) | 187 (26.4) | ||||
Lowest | 190 (13.5) | 185 (14.2) | 157 (14.4) | 209 (14.6) | 199 (14.8) | ||||
Total tocopherol | Highest | 225 (22.9) | 179 (19.2) | 224 (19.0) | 198 (18.6) | 158 (18.3) | 143 (18.2) | ||
Lowest | 205 (6.4) | 208 (6.5) | 292 (6.9) | ||||||
Total tocotrienol | Highest | 287 (36.8) | 236 (31.0) | 276 (30.7) | 163 (30.4) | 201 (30.1) | |||
Lowest | 216 (17.4) | 196 (17.7) | 185 (18.7) | 268 (18.9) | |||||
Total tocols | Highest | 287 (54.9) | 179 (48.7) | 291 (47.5) | 225 (46.9) | 151 (46.1) | 156 (45.9) | 198 (45.9) | |
Lowest | 204 (26.8) | 208 (27.6) | 205 (28.1) | 209 (28.1) | 268 (28.3) | ||||
Total phytosterols | Highest | 225 (320.2) | 187 (315.2) | 287 (285.1) | 290 (283.9) | 293 (281.9) | |||
Lowest | 185 (162.9) | 190 (163.9) | 212 (169.9) | 199 (171.3) | 157 (172.8) |
The squalene contents in 157 Korean-bred rice varieties ranged from 7.9 (RWG 258) to 78.4 μg/g (RWG225) with an average of 35.2 μg/g (Tables 1, 2). Although both japonica-(7.9-78.4 μg/g) and indica-type (8.1-76.4 μg/g) varieties showed similar range of minimum and maximum squalene contents, 2.5 times higher average squalene contents were observed in japonica-type varieties (38.3 μg/g) compared to indica-type (15.2 μg/g) ones. Among 157 accession, highest and lowest brown rice campesterol contents were observed in RWG225 (71.4 μg/g) and RWG165 (27.8 μg/g), respectively and average CA content in 157 accessions was 42.1 μg/g. In the case of stigmasterol, RWG293 (27.7 μg/g) and RWG 190 (13.5 μg/g) recorded the highest and lowest content accessions, respectively and its average content was 20.0 μg/g. Among phytosterols, sitosterol was the major one consisting of over 70% of all phytosterols. Consequently, sitosterol showed relatively higher average content (163.5 μg/g) compared to CA and ST and its minimum and maximum contents were 114.8 μg/g and 235.3 μg/g as observed in RWG190 and RWG293, respectively. Total phytosterols content ranged from 162.9 μg/g (RWG185) to 320.2 μg/g (RWG225). Unlikely to the cases of tocols, both japonica- and indica-type varieties showed similar phytosterols content properties in that japonica-type varieties showed campesterol, sitosterol, stigmasterol, and total phytosterol contents ranging 28.2-71.4, 114.8-235.3, 13.5-27.7, and 162.9-320.2 μg/g, respectively, which were similar to 27.8-60.4, 141.1-198.6, 14.6-22.2, and 192.6-265.2 μg/g observed in indica-type varieties. Histograms of squalene and 3 phytosterols contents in 157 Korean-bred rice varieties are represented in Fig. 2.
The results presented in Table 3 revealed that major fatty acids in brown rice of 157 Korean-bred varieties were linoleic, oleic, palmitic acids consisting 36.5%, 35.8%, and 22.9% of 9 kinds of quantified fatty acids. Besides these major ones, stearic, linolenic, palmitic, arachidic, eicosenoic, and behenic acid showed average compositions of 1.68%, 1.12%, 0.93%, 0.41%, 0.37%, and 0.34%, respectively. These fatty acids compositions resulted in 26.25% of saturated, 36.16% of mono-unsaturated, and 37.61% of polyunsaturated fatty acid in brown rice of 157 Korean-bred rice varieties. Ecotype-dependent difference in fatty acid compositions were observed in that japonica-type 137 varieties showed higher average compositions in palmitic (22.57%), stearic (1.66%), oleic (36.10%), arachidic (0.41%), eicosenoic (0.38%), behenic (0.34%), and total saturated (25.91%) and mono-unsaturated (36.47%) fatty acids compared to indica-type varieties, while no statistical differences were observed in myristic, linoleic, linolenic, and polyunsaturated fatty acids. Histograms for brown rice fatty acid compositions are given in Fig. 3.
Table 3 . Statistics for brown rice fatty acid composition (%) of 136 japonica- and 21 indica-type cultivars bred in South Korea.
Ecotype | Statistics | Myristic (C14:0) | Palmitic (C16:0) | Stearic (C18:0) | Oleic | Linoleic (C18:2) | Linolenic (C18:3) | Arachidic (C20:0) | Eicosenoic (C20:1) | Behenic (C22:0) | Saturated fatty acid | Mono-unsaturated fatty acid | Poly-unsaturated fatty acid |
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Indica | Case | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 | 21 |
Mean | 0.94 | 24.76 | 1.80 | 33.83 | 36.43 | 1.15 | 0.46 | 0.30 | 0.32 | 28.29 | 34.13 | 37.58 | |
SDz) | 0.22 | 1.58 | 0.24 | 1.94 | 1.61 | 0.14 | 0.06 | 0.03 | 0.04 | 1.62 | 1.95 | 1.66 | |
Median | 0.97 | 24.91 | 1.84 | 33.53 | 36.29 | 1.19 | 0.44 | 0.31 | 0.33 | 28.05 | 33.88 | 37.40 | |
Mode | 0.97 | 19.64 | 1.97 | 30.40 | 38.22 | 1.24 | 0.43 | 0.29 | 0.29 | 27.71 | 30.69 | 34.32 | |
Minimum | 0.55 | 19.64 | 1.40 | 30.40 | 33.34 | 0.91 | 0.32 | 0.22 | 0.24 | 23.25 | 30.69 | 34.32 | |
Maximum | 1.37 | 27.90 | 2.41 | 39.32 | 40.12 | 1.34 | 0.59 | 0.36 | 0.37 | 31.52 | 39.63 | 41.46 | |
Range | 0.82 | 8.26 | 1.01 | 8.92 | 6.78 | 0.43 | 0.27 | 0.14 | 0.13 | 8.27 | 8.94 | 7.14 | |
RSDy) (%) | 23.3% | 6.4% | 13.5% | 5.7% | 4.4% | 11.8% | 13.4% | 11.5% | 11.3% | 5.7% | 5.7% | 4.4% | |
Japonica | Case | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 | 136 |
Mean | 0.92 | 22.57 | 1.66 | 36.10 | 36.51 | 1.11 | 0.41 | 0.38 | 0.34 | 25.91 | 36.47 | 37.62 | |
SD | 0.18 | 1.22 | 0.31 | 2.45 | 2.10 | 0.11 | 0.06 | 0.04 | 0.04 | 1.19 | 2.47 | 2.14 | |
Median | 0.90 | 22.66 | 1.66 | 35.79 | 36.70 | 1.10 | 0.39 | 0.37 | 0.34 | 25.99 | 36.16 | 37.84 | |
Mode | 0.84 | 21.92 | 1.70 | 33.52 | 36.00 | 1.07 | 0.39 | 0.37 | 0.32 | 25.26 | 34.66 | 37.02 | |
Minimum | 0.56 | 18.99 | 1.04 | 30.49 | 28.85 | 0.87 | 0.30 | 0.27 | 0.26 | 23.05 | 30.80 | 29.93 | |
Maximum | 1.34 | 25.23 | 2.55 | 44.26 | 41.59 | 1.40 | 0.64 | 0.52 | 0.46 | 28.57 | 44.70 | 42.83 | |
Range | 0.78 | 6.24 | 1.51 | 13.77 | 12.74 | 0.53 | 0.34 | 0.25 | 0.20 | 5.52 | 13.90 | 12.90 | |
RSD (%) | 19.2% | 5.4% | 18.3% | 6.8% | 5.7% | 10.1% | 15.8% | 11.0% | 12.5% | 4.6% | 6.8% | 5.7% | |
Total | Mean | 0.93x) | 22.87* | 1.68* | 35.79* | 36.50x) | 1.12x) | 0.41* | 0.37* | 0.34* | 26.23* | 36.16* | 37.61x) |
SD | 0.18 | 1.47 | 0.30 | 2.50 | 2.04 | 0.12 | 0.07 | 0.05 | 0.04 | 1.49 | 2.53 | 2.08 | |
RSD (%) | 19.7% | 6.4% | 17.9% | 7.0% | 5.6% | 10.3% | 16.0% | 13.0% | 12.5% | 5.7% | 7.0% | 5.5% |
z)Standard deviation.
y)RSD: Relative standard deviation (%).
x)Not significant difference between ecotypes by t-test (
*Significant difference between ecotypes by t-test (
All tested phytonutrients content and fatty acid composition data were used for correlationship (Fig. 4, Supplementary Table S2) and multivariate (Fig. 5) analyses. Positive correlations could be observed between α-T and α-T3 (
Long history of rice cultivation in Korean peninsula provided not only diverse wild and weedy rices but also various traditionally cultivated landraces. Modernized rice breeding programs in Korea started from 1936 have produced more than 600 Korean rice varieties (Cho
In the present study, average contents of α-T, γ-T, α-T3, and γ-T3 were 11.9, 1.6, 10.0, and 13.9 μg/g, respectively. Although another form of tocols such as β-T, δ-T, β-T3 were also detected and quantified in our experiments, their average contents were as low as 0.69, 0.60, and 0.28 μg/g, respectively (data not shown) so that their data were provided in this report considering their relatively low content and subsequent low importance in health-related effects. These findings are quite similar to previous reports (Huang
Unlikely to the case tocopherols, studies on tocotrienol form of vitamin E are limited, and especially in the case biosynthetic pathway of tocotrienols it is assumed that same genes involved in tocopherol synthesis may also work for tocotrienol synthesis (Lu
Among tested 157 varieties, squalene content ranged from 7.9 to 78.4 μg/g with an average of 35.2 μg/g, which was similar to previous report of Wang
The 157 Korean-bred varieties used in this experiment consisted of 13 varieties developed from 1930’s to 1969, and 15, 24, 41, 50, and 14 varieties developed in 1970’s, 1980’s, 1990’s, 2000’s and 2010’s, respectively. When any trends in variety development year-dependent changing patterns in phytonutrient contents were evaluated, it was found that most of tested phytonutrients were rarely affected by development decades (Supplementary Table S3). Exceptional cases were the rice varieties developed in 1970’s, which showed relatively lower contents of α-T (8.7 μg/g) and α-T3 (6.8 μg/g) but relatively high γ-T (3.1 μg/g) and γ-T3 (18.0 μg/g) compared to varieties developed in an-other decades. These findings, however, is deeply related with the fact that 1970’s was the decade when indica-type high yield rice variety were highly developed and released. As already described, indica-type rice contains lower α-T and α-T3 but higher γ-T and γ-T3 (Table 1). Consequently, abovementioned variety development decade-dependent changes in phytonutrients are deeply related with ecotypes of varieties mainly developed at each decade.
Among 3 major fatty acids, palmitic and stearic acids and consequently saturated and mono-unsaturated fatty acid compositions were lower in japonica-type varieties compared to indica-type ones (Table 3) and these results were consistent previous report (Goffman
Application of multivariate analysis such as PLS-DA revealed that profiles of phytonutrients can be used for differentiation of rice ecotypes (Fig. 5), and α-T3/γ-T3, α-T, α-T3, α-T, and γ-T were the major components in determining ecotype of rice based upon their high VIP scores (Fig. 5B). Exceptional two cases were RWG164 and RWG262, which exhibited phytonutrient properties more similar to japonica- and indica-type, even though whose ecotypes are indica- and japonica-, respectively. Further studies on why RWG164, an indica-type accession could have high α-T content as well as high α-T/γ-T and α-T3/γ-T3 ratios similar to the levels of japonica-type varieties seems to be required.
In this report, genetic variations in tocols, squalene, phytosterols contents and fatty acid compositions in brown rice of Korean-bred 157 rice varieties were presented together with a list of varieties containing exceptionally high or low quantity of each phytonutrient. These findings may serve as genetic resource information for breeding a superior rice variety with higher nutritional value.
This work was supported by a grant from the Next-Generation BioGreen 21 Program (Plant Molecular Breeding Center No. PJ013280), Rural Development Administration, Republic of Korea and the Research Fund of Soonchunhyang University, Asan, Republic of Korea.
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