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Genome-wide association mapping of polyphenol contents and antioxidant capacity in whole grain rice Feifei Xu, Jinsong Bao, Tae-sung Kim, and Yong-Jin Park J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b01289 • Publication Date (Web): 10 May 2016 Downloaded from http://pubs.acs.org on May 14, 2016

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Journal of Agricultural and Food Chemistry

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Genome-wide association mapping of polyphenol contents and

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antioxidant capacity in whole grain rice

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Feifei Xu†, ‡, ||, Jinsong Bao†, ‡, ||, Tae-Sung Kim† and Yong-Jin Park*, †,

§

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†

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University, Yesan, Republic of Korea

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‡

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Zhejiang University, Huajiachi Campus, Hangzhou, 310029, China

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§

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Department of Plant Resources, College of Industrial Science, Kongju National

Institute of Nuclear Agricultural Science, College of Agriculture and Biotechnology,

Legume Bio-Resource Center of Green Manure (LBRCGM), Kongju National

University, Yesan, Republic of Korea

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|| These authors equally contribute to this work

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* To whom correspondence should be addressed. E-mail:[email protected];

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Tel: +82 41-330-1201; Fax: +82- 41-330-1209

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ACS Paragon Plus Environment


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ABSTRACT

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Whole grains contain various bioactive phytochemicals including phenolic acids, and

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consumption of whole grain may provide desirable health benefits and reduce the

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risks of chronic diseases due to their antioxidant activities. In this study, we qualified

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and quantified 9 bound phenolic compounds in 32 red and 88 white pericarp

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accessions of rice. Genome-wide association study (GWAS) was conducted for free

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(FP) and bound (BP) phenolic compounds and their antioxidant capacities with

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high-quality single nucleotide polymorphisms (SNPs) in two colored grain panels and

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the whole panel. Rc was detected for all FP and antioxidant capacities in the whole

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panel. Three loci (chr.1:30970095, chr.6: 24392269 and chr.9: 6670223) for more than

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five phenolic-related traits, two loci (chr.4: 34120529 and chr.11: 28947480) for more

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than two FP-related traits, and one locus (chr.11: 23220681) for ferulic acid detected

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in two panels were potentially new genes that are valuable for further gene cloning.

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Overall, this study increases our understanding on the genetics of phenolic acid

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biosynthesis in the phenylpropanoid pathway.

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KEYWORDS: Polyphenol content, antioxidant capacity, phenolic acid, GWAS, rice

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INTRODUCTION

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Whole grain cereals are known as functional foods or nutraceuticals, which can

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reduce the risk of coronary heart disease,1 insulin resistance, and type-2 diabetes2 due

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to the antioxidant capacities of phytochemicals present in the grains. Therefore,

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consumers are interested in healthy diets of whole grain foods, and breeders have

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attempted to breed new varieties with high antioxidant activities.

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Pigmented whole grain rice has several colors, ranging from white to red to black

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(purple),3 which is determined based on the composition of anthocyanin pigments that

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are ubiquitous throughout the plant kingdom.4 The majority of antioxidant compounds

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are mainly distributed in bran (pericarp, seed coat, nucellus, and aleurone), followed

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by the embryo, and a small amount is detected in the endosperm.1, 5-7 In general,

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antioxidant capacity is strongly correlated with pericarp color; black and red rice

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varieties exhibit higher antioxidant activities than white rice varieties due to the

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presence of anthocyanins in black rice and proanthocyanidins in red rice.8-11 However,

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some study showed that in red and black rice cultivars, anthocyanin pigments

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contributed little to the total antioxidant capacity, and other phenolic compounds

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contributed a lot to their antioxidant capacity.4 Besides anthocyanins and

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proanthocyanidins, phenolic acids play a role in determining antioxidant activities in

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white rice varieties. It seems that phenolic acids are mainly in the form of insoluble

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forms, while flavonoids and anthocyanins are mainly in soluble forms.8, 12 Ferulic acid

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(56~77% of total phenolic acids) is the most abundant phenolic acid that mainly

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existed in the endosperm, bran, and whole grain, followed by p-coumaric (8~24%),

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sinapic (2~12%), gallic (1~6%), protocatechuic (1~4%), p-hydroxybenzoic (1~2%),

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vanillic (1%), and syringic acids (1%). While caffeic acid, chlorogenic acid, cinnamic

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acid, and ellagic acid are minor constitutes that account for less than 1% of total 3



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phenolic acids.8 Phenolic compounds and antioxidant properties in whole grain rice

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vary with the seed development stages,13 varieties,14 and grain shape.15 Despite

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detailed information on the structure, component, distribution, and dynamic changes

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in polyphenols, only a few studies have been reported regarding the genetics of FP

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and BP compounds, as well as the antioxidant capacities of whole grain.

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Two genes affecting proanthocyanidin synthesis in red- and brown-colored rice

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were elucidated.16, 17 The Rc gene encodes a basic helix-loop-helix (bHLH) protein,

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and the recessive white rice allele rc has a 14-bp deletion within exon 6 that knocks

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out the bHLH domain of the protein, resulting in the white color. Rd, encoding

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dihydroflavonol-4-reductase, is a gene related to anthocyanin biosynthesis. Rc, in the

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absence of Rd, produces brown seeds, whereas Rd alone has no phenotype. One gene

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regulating anthocyanin synthesis in black-colored rice was characterized;18 the black

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grain trait originated from ectopic expression of the Kala4 bHLH gene due to a

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rearrangement in the promoter region. Both Rc and Kala4 genes activate upstream or

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downstream flavonol biosynthesis genes to produce specific pigments. Kim et al.

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investigated the expression of five anthocyanin biosynthetic genes and showed that

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their expression was enhanced during seed maturation and correlated with ambient

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temperature during seedling growth. Based on association mapping, Shao et al.

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identified 41 marker loci for grain color, phenolic content, flavonoid content, and

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antioxidant capacity, and confirmed that Ra and Rc genes are main-effect loci for rice

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grain color and polyphenol traits.20 The RM316 locus, located on chromosome 9, has

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a significant association with phenolic content. Shao et al. evaluated the genotype ×

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environment interactions for polyphenols and antioxidant capacity of rice, 3 finding

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that genotype dominantly controls polyphenol and antioxidant capacities with > 94%

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phenotype variation. They also detected 23 quantitative trait loci (QTLs); two QTLs 4


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located close to Ra and Rc were detected in both years, and others that were sensitive

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to the environment were detected only in one year. However, excluding Rc and Rd, all

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QTLs for polyphenols were localized at a large interval, and high-resolution

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resequencing data is required for the detection of new QTLs for polyphenols.

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Genome-wide association studies (GWAS) is a comprehensive approach that can

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systemically search for causal genetic variation inrice genome,21 and succeed in

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detecting QTLs for grain weight,22 agronomic traits, 23 polyphenol contents,3 among

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others. However, identification of QTLs for phenolic acids, such as ferulic acid,

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p-coumaric acid, and sinapic acid, etc. has not been reported.

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The objective of the present investigation was to (1) identify and quantify bound

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phenolic acids in whole grain rice and (2) detect QTLs for TPC, FPC, TFC, and BPC

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compounds, as well as their antioxidant capacities in three panels, including the red

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grain panel, white grain panel, and whole panel (red grain panel plus white grain

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panel).

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MATERIALS AND METHODS

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Plant Materials. A core collection of 137 accessions was established previously

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based on SSR markers using a heuristic approach with PowerCore software.24,

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Among them, 120 accessions, including 88 white pericarp (white grain panel) and 32

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red pericarp accessions (red grain panel), were used in this study. The whole panel

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was planted in 2014 during the growing season from late April to late September at

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Kongju National University farm, Yeasan Gun, South Korea. The grains were

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air-dried to a moisture content of about 12%, stored at room temperature for 3 months.

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The samples were de-hulled into brown rice, and then ground and passed through a

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100-mesh sieve to obtain whole grain flour. All the flour samples were stored at 4 °C 5


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prior to extraction.

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Extraction of Soluble-free Phenolic Compounds. The extraction of soluble-free

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phenolic compounds in whole grain materials was performed according to the method

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described by Yu et al. 26， with minor modifications. Briefly, 0.5 g of whole grain flour

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was extracted twice with 5 mL of 80% methanol. After shaking at 250 rpm for 1 h

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using an oscillator (HZ- 9210K Desktop frozen oscillator, Jiangsu, China), the

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mixture was centrifuged at 3000rpmfor 10 min. The supernatants of each duplicate

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extraction were collected and combined together, and then stored at −20°C until use.

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Duplicate extractions were performed for each sample.

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Extraction of Insoluble-bound Phenolic Compounds. Bound phenolics extraction

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was performed as described by Yu et al.

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Residue obtained from the crude extraction was washed twice with 20 mL of ddH2O

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to eliminate organic solvent and then subjected to alkaline hydrolysis with 20mL of

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4M NaOH on a shaker (HZ-9210K Desktop frozen oscillator, Jiangsu, China) (250

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rpm) for 2 h. The resultant mixture was adjusted to a pH between 1.5 and 2.0 with 6 N

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HCl, and then extracted with 40mLof ethyl acetate three times (20, 10, and 10mL).

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The supernatant was collected and combined into an Erlenmeyer flask and then

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evaporated to dryness at 35°C under vacuum using a rotary evaporator and water bath

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(RE-2000A, Ya Rong Biochemistry Instrument Factory, Shanghai, China). The dried

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residue was re-dissolved in 2 mL of 50% methanol and stored at -20°C. Prior to

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HPLC analysis, they were filtered through a 0.45-µm syringe filter.

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and Qiu et al.10, with minor modifications.

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Determination of Total Phenolic Content (TPC). Folin-Ciocalteu assay was 6


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performed to determine TPC in the soluble-free (FPC) and insoluble-bound (BPC)

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fractions according to Shao et al.3 First, 10-fold-diluted Folin-Ciocalteu reagent and

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appropriately diluted samples (red rice free phenolics: 6-fold-diluted; all bound

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phenolics: 5-fold-diluted, and white rice free phenolics: not diluted) were prepared

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prior to measurements. Briefly, 200 µL of sample extracts or standards solution was

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mixed completely with 1.5 mL of Folin-Ciocalteu reagent and reacted for 5 min. Next,

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1.5 mL of saturated sodium carbonate (75 g/L) was added to the mixture. After 2 h in

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the dark at room temperature, the absorbance of the resulting blue color was recorded

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using a spectrophotometer at 725 nm. Duplicate determinations were conducted for

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each extract. The calibration curve was prepared using gallic acid solutions. Phenolic

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content is expressed as milligrams of gallic acid equivalent per 100 g (mg GAE/100g)

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of dry weight whole grain rice flour.

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Determination of Total Flavonoid Content (TFC). Soluble-free phenolic extracts

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were used to determine the TFC. Solution A (5% NaNO2), solution B

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(10%AlCl3·6H2O), and 1M NaOH was prepared before measurements.3 First, 150 µL

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of solution A was mixed completely with 1mL of appropriately diluted samples or

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standards solution and reacted for 5 min. Second, 150 µL of solution B was mixed

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completely with the above mixture and reacted for 5 min. Third, 1mL of 1M NaOH

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solution was added to the mixture, after which 3 mL of ddH2O was immediately

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added to the mixture. Finally, after 45 min in the dark at room temperature, the

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absorbance of the resulting yellow color was recorded using a spectrophotometer at

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510 nm. TFC is expressed as milligrams of catechin acid equivalent per 100 g of rice

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flour (mg CAE/100 g).

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Determination of ABTS·+(2, 2-azino-bis-(3-ehylbenzothiazoline-6-sulphonic acid)

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diammonium salt) Radical Scavenging Activity. The antioxidant capacities of rice

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extracts

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spectrophotometer based on the improved ABTS radical cation color decolorization

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assay27 with minor modifications.3 The working solution of ABTS was prepared by

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mixing 7 mM ABTS and 2.45 mM potassium persulfate (v/v, 2:1) and reacted in the

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dark at room temperature for 12~16 h before use. The ABTS•+ working solution was

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diluted in methanol to adjust the absorbance to 0.700 ± 0.020 at 734 nm. Then, 3.9 mL

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of ABTS•+ working solution was completely mixed with 100 µL of sample or

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standards and reacted in the dark at room temperature for 45 min. Subsequently, the

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absorbance was recorded using a spectrophotometer at 734 nm. Results are expressed

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as µmol of Trolox equivalent antioxidant capacity per 100 g of dry weight rice flour

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(µmol TE/100 g).

(free

phenolic

and

bound

phenolic)

were

determined

using

a

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Determination of DPPH (2, 2-diphenyl-1-picrylhydrazyl) Radical Scavenging

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Activity. Free phenolic and bound phenolic antioxidant capacities were also

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determined using the DPPH assay.3 Briefly, 3 mL of 100 µM DPPH solution was

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completely mixed with 200 µL of sample or standard solution and reacted in the dark

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at room temperature for 45 min. The absorbance was recorded using a

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spectrophotometer at 517 nm. The DPPH scavenging activity (%) of both samples and

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standard (Trolox) was calculated, as follows:

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DPPH (%)= (1-Asample/Acontrol) ×100 The results are expressed as Trolox equivalent DPPH radical scavenging activity per 100 g of sample (µmol TE/100 g).

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Identification and Quantification of Phenolic Acids with High Performance

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Liquid Chromatography (HPLC). Identification and quantification of bound

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phenolic compounds were performed on an HPLC (Waters 2695, Milford, MA)

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equipped with a photodiode array (PDA) detector (Waters 2998) and an autosampler

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(Waters 717 plus) according to the method described by Yu et al. 26 and Shen et al. 28.

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A column with the following specifications, 150 mm × 4.6 mm, 5 µm C18 110 A

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column (Phenomenex, Torrance, CA), was used for HPLC separation. The

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temperature was set at 35°C and the injection volume was 10 µL using an auto

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sampler. The mobile phase consisted of A (0.1% acetic acid in water) and B (0.1%

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acetic acid in methanol), and the flow rate was 0.9 mL/min. A 70 min linear gradient

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was set as follows: 0~11 min, 9~14% B, 11–14 min, 14~15% B, 14~17 min, 15% B,

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17~24 min, 15~16.5% B, 24~28 min, 16.5~19% B, 28~30 min, 19~25% B, 30~36

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min, 25~ 26% B, 36~38 min, 26~28% B, 38~41 min, 28~35% B, 41~46 min, 35~40%

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B, 46~48 min, 40~48% B, 48~53 min, 48~53% B, 53~65 min, 53~70% B, 65~66 min,

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70~9% B, 66~70 min, and 9% B. Phenolic acids were detected at a wavelength of 280

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nm. A total of 11 phenolic acid standards were used in this study (Figure S1),

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corresponding to gallic acid, protocatechuic, p-hydroxybenzoic acid, vanillic acid,

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caffeic acid, syringic acid, p-coumaric acid, ferulic acid, sinapic acid, iso-ferulic acid,

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and o-coumaric, respectively. The contents of phenolic acids were quantified based on

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the standard curves of the corresponding phenolic acids at the wavelength of 280 nm.

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Statistical Analysis and Genome-wide Association Mapping. All statistical

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analyses were performed with SAS program version 9.1 (SAS Institute Inc, Cary, NC,

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USA). Proc Corr was used to examine Pearson correlation between different traits,

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and the soluble-free phenolic-related traits and insoluble-bound phenolic-related traits 9



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were performed separately. Analysis of variance (ANOVA) was performed using the

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general linear model procedure (Proc glm), and Duncan’s new multiple range test was

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used to determine significant differences between red and white grain rice panels.

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Genome resequencing data of the 120 accessions (whole panel) were collected

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from the 295 rice whole genome resequencing data, which were obtained previously

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using HiSeq 2000 and HiSeq 2500.29 To control for false-positives, the P＋K model

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performed based on the mixed linear model (MLM) was used for genome-wide

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association mapping of phenolic acid-related traits in three panels by GAPIT.30 SNP

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sites with the lowest P value in the peak region (P

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