Edited by: Thomas M. Davis, University of New Hampshire, United States
Reviewed by: Dongying Gao, University of Georgia, United States; Kanako Bessho-Uehara, Carnegie Institution for Science (CIS), United States
†These authors have contributed equally to this work
This article was submitted to Plant Breeding, a section of the journal Frontiers in Plant Science
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The pericarp color of rice grains is an important agronomic trait affected by domestication, and the color pigment, anthocyanin, is one of the key determinants of rice nutritional quality. Weedy rice, also called red rice because its pericarp is often red, may be a novel gene resource for the development of new rice. However, the genetic basis and nutritional quality of anthocyanin are poorly known. In this study, we used a genome-wide association study (GWAS) to find novel and specific QTLs of red pericarp in weedy rice. The known key gene site of red pericarp
Weedy rice (
In general, the morphological characteristics of weedy rice fall between the wild rice species (
Brown rice refers to the caryopsis after the rice husk is removed, and the pericarp, seed coat, and nucellus are intact. Milled rice refers to rice grains with pericarps and seed coats removed. Colored rice such as red rice, gold rice, and black rice, refers to brown rice with colored pericarp (
With recent developments in genetic analysis, some molecular mechanisms regulating rice pigmentation are well known. For instance the red color of pericarp is affected by the interaction of two genes,
The pericarp color is an important agronomic trait affected by domestication, and the color pigment, anthocyanin, is one of the important determinants of rice nutritional quality (
We collected 297 rice samples of six subgroups, including 46 weedy rice from Asian high latitudes (
In order to further understand the production and distribution of weedy rice pericarp pigments, the typical red pericarp weedy rice WR07-14, WR07-47, WR03-32, WR07-141, WR07-142 and WR03-29 and white pericarp cultivated rice Shennong265, Akihikari, Nipponbare, Qishanzhan were selected from a GWAS panel for the observation of pigment components and their deposition process and for a nutrient quality analysis.
For GWAS, the phenotype value of pericarp color was defined as one of four levels: 0 for white, 1 for orange, 2 for red, 3 for dark red. Short Oligonucleotide Alignment Program (SOAP) software (
In the present studies, we performed imputation to fill the missing genotype for the 122,777 unimputed SNPs by using the k-nearest neighbor algorithm (KNN) model in two sets of GWAS populations (
In this study, red pericarp rice (including landrace and weedy rice) were considered as the unimproved rice population, and modern cultivars with white pericarp were considered as the improved population. Nucleotide diversity (π) and Tajima’s
The weedy rice that we used to observe the development of pericarp color were sampled every 2 days after flowering. The caryopsis to be used for hand-sliced sections were sampled every week after flowering. A digital SLR camera (Canon 550D) was used to record pericarp color and pigment distribution (
We further sampled the pericarp, seed coat, and endosperm of weedy rice 1, 2, and 3 weeks after flowering respectively in order to study the distributions of their pigments. Referring to the method of
We collected seeds of each sample (WR07-14, WR07-32, WR07-47 and WR07-141) at 5, 10, 15, 20, and 25 days after flowering, and we threshed, shelled, and ground the rice into flour. The total content of anthocyanins was determined by the pH differential method (
Metal element concentrations in the pericarp of weedy rice and control samples were measured by using inorganic mass spectrometry (
Statistical analyses for the comparison of phenotypic values ANOVA were carried out with the statistical software IBM SPSS 2.00 (IBM Crop, Armonk, NY, United States). The level of significance taken as
The red pericarp phenotype was observed in all subgroups (
Pericarp color grading statistics. Each circle represents a subcategory of rice, and the area occupied by each color represents the proportion of rice seeds of each pericarp color.
GWAS results presented as Manhattan plot for
GWAS peak selection sweep and candidate genes.
Chr1:12490628 | 4.69E−11 | 12316796-12821546 | 2.12 | 3.21 | –0.26 | Weak | ||
Chr1:24954656 | 1.3E−09 | 24900449-25222998 | 1.87 | 3.11 | 0.09 | Weak | LOC_Os01g43700.1( |
Cytochrome P450 72A1, putative, expressed |
Chr1:33719449 | 5.7E−11 | 33334269-33780964 | 2.42 | 3.04 | –0.78 | Weak | LOC_Os01g58950.1 | Cytochrome P450, putative, expressed |
Chr3:4655686 | 1.38E−11 | 4045839-4658179 | 2.78 | 2.6 | –1.7 | Strong | LOC_Os03g08930 | Basic helix-loop-helix dimerisation region bHLH domain containing protein; |
Chr3:9158937 | 1.12E−07 | 9326824-9810369 | 3.48 | 2.8 | –0.79 | Strong | ||
Chr3:13155280 | 3.11E−12 | 12620491-13044531 | 3.83 | 1.88 | –1.45 | Strong | ||
Chr4:12679715 | 3.22E−07 | 12320456-13135109 | 2.39 | 2.41 | –0.97 | Weak | ||
Chr4:20685858 | 1.82E−09 | 21337500-21848954 | 2.4 | 0.9 | –1.3 | Weak | ||
Chr6:8148068 | 3.41E−09 | 7070518-7601689 | 3.20 | 3.1 | –1.2 | Strong | ||
Chr7:6197350 | 4.88E−12 | 5536887-6172772 | 4.09 | 1.44 | –0.78 | Strong | LOC_Os07g11020 ( |
Pericarp color; red pericarp |
Chr9:656027 | 6.98E−10 | 6084684-6583948 | 2.62 | 1.72 | –1.46 | Strong | ||
Chr10:296002 | 8.23E−11 | 2605942-3053511 | 1.61 | 1.94 | –0.19 | Weak | ||
Chr12:23036595 | 1.23E−13 | 22936893-23280883 | 3.16 | 0.56 | –1.76 | Strong | LOC_Os12g37419 | Cytochrome c oxidase polypeptide Vc, putative, expressed 6 |
Selection sweep analyses were conducted in the “unimproved” red pericarp population and in the “improved” cultivated white pericarp population. The selection sweep parameter of πunimproved population/πimproved population per 500 kb genomic slide windows were highlighted from yellow (0.17) to magenta (4.22) (
Selection sweep analysis and candidate genes for weedy rice pericarp color.
The typical red pericarp weedy rice WR07-14, WR07-47, WR03-32, WR07-141, were selected from the GWAS panel for the observation of pigment components and the deposition process. The pericarp color of all sampled weedy rice was green in the early stage of development (0–5 days after flowering). The pigment began to deposit on both ends of the caryopsis (6–8 days after flowering), accumulating along the vascular bundle at the back of the caryopsis (9–15 days after flowering), and finally occurring throughout the entire caryopsis (16–18 days after flowering). When the seeds were mature, the pigmented pericarps were slightly faded (26–28 days after flowering) (
Changes through time in the appearance of the pericarp and pigmentation sites of typical weedy rice.
We dissected the weedy rice WR07-14 caryopsis, including pericarp, seed coat, and endosperm (including aleurone layer) at different stages of development. At 1–7 days after flowering, the caryopsis was green, the seed coat was transparent, and the endosperm was white. At 8–13 days after flowering, the accumulated pigment in the pericarp was a light color, the thin seed coat was lighter in color than the pericarp, and the endosperm was still white. At 14–21 days after flowering, the pericarp presented dark red, the seed coat was colored but thinner than the pericarp, and the endosperm was white. Based on these observations, the pigment was mainly deposited in the pericarp, a small amount was deposited in the seed coat, and the pigment was absent in the endosperm (
At 2 weeks after flowering, the pericarp and seed coat cells of the weedy rice began to shrink, and the binding between them gradually became tight. The pericarp cells began to shrink, and the pigment began to accumulate mainly in the lower part of the mesocarpal cells. A small amount of pigment was deposited in the outer pericarp. Pigments were difficult to detect in the seed coat. The aleurone cells began to form out of the outer layer of endosperm, and they arranged themselves into a rectangular shape. At 3 weeks after flowering, the cell binding between pericarp and seed coat was more compact, and the seed coat layer was completely flattened. A large amount of pigment was deposited in the pericarp, and a small amount was deposited in the seed coat. The gap between the aleurone layer and the endosperm cells shrank, and neither the aleurone layer nor the endosperm portion was pigmented (
To determine changes in pericarp pigmentation with weedy rice seed growth, we further measured the changes of anthocyanin concentration and composition in four typical weedy rice accessions, WR07-14, WR07-47, WR03-32, and WR07-141, since the anthocyanin is the main component of pericarp pigment. The concentration of anthocyanins rose and then fell until it stabilized, with the peak appearing sometime between the 15th to 20th day after flowering (
Changes in anthocyanin content in differently colored varieties of weedy rice through different developmental stages.
The anthocyanin components of weedy rice were detected by using the hydrochloric acid-vanillin method and high performance liquid chromatography-mass spectrometry (HPLC) (
Anthocyanin content in different colored weedy rice varieties.
WR07-14 | Dark red | 44.92 ± 0.89a | 5.18 ± 0.12a |
WR07-141 | Red | 5.21 ± 0.22d | N |
WR03-32 | Dark red | 13.9 ± 0.31c | N |
WR07-47 | Dark red | 37.4 ± 0.64b | 5.21 ± 0.29a |
Cultivated red rice is considered to be a beneficial, healthful food (
Metal element content in different rice varieties.
WR07-47 | Dark red | 42.31 | 261.60 | 765.50 | 43.40 | 3.94 | 129.69 | 0.73 | 0.07 | 0.014 |
WR03-32 | Dark red | 23.18 | 164.40 | 762.78 | 28.56 | 2.52 | 59.91 | 0.77 | 0.03 | 0.0068 |
WR03-29 | Red | 26.71 | 165.90 | 785.04 | 24.46 | 2.23 | 48.78 | 0.58 | 0.02 | 0.0008 |
WR07-141 | Red | 38.14 | 177.90 | 986.19 | 29.30 | 3.78 | 73.18 | 1.10 | 0.06 | 0.014 |
WR07-142 | Red | 20.85 | 127.25 | 1048.90 | 28.91 | 3.28 | 45.77 | 0.46 | 0.05 | 0.0069 |
AKI | White | 9.86 | 55.35 | 458.34 | 11.66 | 1.32 | 16.99 | 0.21 | 0.02 | 0.0034 |
SN265 | White | 9.17 | 83.40 | 435.40 | 14.89 | 1.43 | 22.50 | 0.20 | 0.01 | 0.0016 |
QSZ | White | 8.84 | 77.60 | 534.30 | 10.98 | 1.12 | 19.83 | 0.31 | 0.01 | 0.0028 |
NIP | White | 11.37 | 84.00 | 654.60 | 12.87 | 1.44 | 26.01 | 0.22 | 0.02 | 0.0032 |
WR-AVE | Red | 30.24aA | 179.41aA | 869.68aA | 30.93aA | 3.15aA | 71.47aA | 0.73aA | 0.05aA | 0.0085aA |
CR-AVE | White | 9.81bB | 75.09bB | 520.66bB | 12.60bB | 1.33bB | 21.33bB | 0.23bB | 0.015bB | 0.0028bB |
Free amino acids are important biologically active molecules which play a role in the synthesis of proteins and in providing energy for the body and brain activity (
Free amino acid content in different rice varieties.
Asp | 7.27 | 5.58 | 5.87 | 6.54 | 6.074 | 4.85 | 4.21 | 4.12 | 3.95 | 6.27aA | 4.28bB |
Thr | 2.59 | 1.9 | 2.01 | 2.34 | 2.15 | 1.79 | 1.66 | 1.68 | 1.89 | 2.20aA | 1.76bA |
Ser | 3.76 | 2.71 | 2.71 | 2.71 | 2.71 | 2.71 | 2.68 | 2.59 | 2.71 | 2.92aA | 2.67aA |
Glu | 13.39 | 12.66 | 10.67 | 12.25 | 11.04 | 9.43 | 9.6 | 9.23 | 9.68 | 12.0aA | 9.49bB |
Gly | 3.22 | 2.35 | 2.56 | 2.76 | 2.59 | 3.69 | 3.87 | 3.63 | 3.58 | 2.70bB | 3.69aA |
Ala | 4.05 | 2.81 | 3.07 | 3.61 | 3.23 | 3.27 | 3.3 | 3.33 | 3.24 | 3.35aA | 3.29aA |
Cys | 2.84 | 2.65 | 2.65 | 2.69 | 2.85 | 3.49 | 3.59 | 3.36 | 3.61 | 2.73bB | 3.51aA |
Val | 5.88 | 6.3 | 6.89 | 5.59 | 5.63 | 5.21 | 5.13 | 5.22 | 5.19 | 6.06aA | 5.19bA |
Met | 1.96 | 1.612 | 1.793 | 1.82 | 1.78 | 1.8 | 1.83 | 1.78 | 1.67 | 1.79aA | 1.77aA |
Ile | 2.28 | 1.49 | 1.78 | 2.13 | 1.89 | 1.65 | 1.63 | 1.75 | 1.55 | 1.91aA | 1.65aA |
Leu | 5.03 | 3.702 | 3.77 | 4.62 | 4.077 | 3.63 | 3.67 | 3.55 | 3.48 | 4.24aA | 3.58aA |
Tyr | 2.25 | 1.34 | 1.58 | 1.97 | 1.74 | 1.43 | 1.48 | 1.33 | 1.49 | 1.78aA | 1.43aA |
Phe | 5.27 | 4.68 | 5.06 | 5.18 | 5.23 | 4.97 | 5.03 | 4.86 | 4.94 | 5.08aA | 4.95aA |
Lys | 3.1 | 2.41 | 2.55 | 2.89 | 2.85 | 2.5 | 2.55 | 2.45 | 2.41 | 2.76aA | 2.48aA |
His | 1.54 | 0.97 | 0.97 | 1.33 | 1.2 | 0.99 | 1.02 | 0.93 | 0.93 | 1.20aA | 0.97aA |
Arg | 6.35 | 4.53 | 4.81 | 5.73 | 5.26 | 4.79 | 4.73 | 4.89 | 4.67 | 5.34aA | 4.77aA |
NH3 | 0.23 | 0.19 | 0.2 | 0.18 | 0.21 | 0.19 | 0.2 | 0.22 | 0.18 | 0.20aA | 0.20aA |
Total | 71.03 | 57.86 | 59.13 | 64.93 | 60.77 | 56.6 | 55.42 | 56 | 55.4 | 62.64aA | 55.87bA |
Saturated fatty acids (palmitic acid and stearic acid) and unsaturated fatty acids (oleic acid and linoleic acid) were detected in weedy rice and cultivated rice in this study. We found that the overall ratio of saturated to unsaturated fatty acids in the weedy rice was slightly lower than that in cultivated rice. The ratios of saturated fatty acid and unsaturated fatty acid content were 20.53 and 79.47% in weedy rice, and the ratios in cultivated rice is 23.03 and 76.97%. There was no significant difference in the proportion of palmitic acid (a saturated fatty acid) between weedy rice and cultivated rice. The proportion of stearic acid (a saturated fatty acid) in weedy rice was significantly lower than that of control cultivated rice (
Fatty acid content in different rice varieties.
WR07-47 | 20.72 | 1.86 | 36.97 | 40.45 | 100.00 |
WR03-32 | 19.49 | 1.97 | 44.05 | 34.49 | 100.00 |
WR03-29 | 19.69 | 1.81 | 42.86 | 35.60 | 100.00 |
WR07-141 | 17.41 | 1.28 | 42.63 | 38.69 | 100.00 |
WR07-142 | 17.06 | 1.38 | 43.53 | 38.03 | 100.00 |
AKI | 20.11 | 2.81 | 41.59 | 35.49 | 100.00 |
SN265 | 20.22 | 2.71 | 41.55 | 35.52 | 100.00 |
QSZ | 20.48 | 2.80 | 41.49 | 35.23 | 100.00 |
NIP | 20.15 | 2.85 | 41.54 | 35.46 | 100.00 |
WR-AVE | 18.874aA | 1.66aA | 42.01aA | 37.45aA | 100.00 |
CR-AVE | 20.24aA | 2.79bB | 41.54aA | 35.42aA | 100.00 |
Our research has shown certain differences in the development of pigmentation in weedy rice compared to previous studies.
It has been reported that anthocyanin accumulation was positively correlated with Superoxide Dismutase (SOD) in seeds, and anthocyanin accumulation led to darker seed coat color (
During the domestication of
In this study, the association signals of purple rice genes
Rice anthocyanins could be a beneficial part of human diets due to their high antioxidant activities. Lei pointed out that red rice pigment was synthesized and deposited only in the pericarp of the caryopsis and absent in the seed coat and aleurone layer (
Rice nutritional quality has attracted more attention in the traditional growing areas of Asia, where monotonous consumption of rice may lead to deficiencies of essential trace elements, amino acid, and other nutrients (
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.
DM, JS, and MZ conceived the project and experiment. JS, WW, and MZ performed the SLAF sequencing and population genetic analysis. WW observed the process of pigmentation in weedy rice and cultivated rice. WW and JyS examined the composition and content of pigment in weedy rice and cultivated rice, and analyzed the nutritional quality of weedy rice and cultivated rice. WW, JS, DM, MZ, GZ, ZL, YH, and XJ provided the germplasm and performed the germplasm management. JS and WW conducted a selection strength analysis. WW, JS, MZ, and DM interpreted the data and wrote the manuscript. All authors contributed to the manuscript and approved the submitted version.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
The Supplementary Material for this article can be found online at:
QQ Plot diagram of the improved and unimproved red rice populations’ GWAS results.
Genome-wide selection sweep with GWAS peak.
Material information of the GWAS populations.
cyanidin-3-glucoside
genetically-modified organism
genome-wide association study
k-nearest neighbor algorithm
paeoniflorin-3-glucoside
quantitative trait locus
weedy rice
weedy rice from Asian high latitudes
weedy rice from south china.