Relationships among Genetic, Structural, and Functional Properties of

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On the relationships among genetic, structural and functional properties of rice starch Xiangli Kong, Yaling Chen, Ping Zhu, Zhongquan Sui, Harold Corke, and Jinsong Bao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.5b02143 • Publication Date (Web): 17 Jun 2015 Downloaded from http://pubs.acs.org on June 24, 2015

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Journal of Agricultural and Food Chemistry

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On the relationships among genetic, structural and functional

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properties of rice starch

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Xiangli Konga, Yaling Chena, Ping Zhua, Zhongquan Suib,*,Harold Corkec,d,

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Jinsong Baoa,*

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a

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Zhejiang University, Hangzhou, 310029, China

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b

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Shanghai Jiao Tong University, Shanghai, 200240, China

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

Department of Food Science and Engineering, School of Agriculture and Biology,

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c

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Kong, China

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d

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Technology, Wuhan, 430068, China

School of Biological Sciences, The University of Hong Kong, Pokfulam Road, Hong

Glyn O. Philips Hydrocolloid Research Centre at HUT, Hubei University of

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*

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Zhongquan Sui, Tel: +86-21-34206613, Fax: +86-21-34206613, Email:

16 17 18

Corresponding authors:

[email protected]; Jinsong Bao, Tel: +86-571-86971932; Fax: +86-571-86971421E-mail: [email protected].

19 20

Abbreviations: AAC, apparent amylose content; CLap, average chain-length of

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amylopectin; BD, breakdown viscosity; CPV, cool paste viscosity; DP, degree of

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polymerization; GPC, gel permeation chromatography;

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anion-exchange

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chromatography; PTemp, pasting temperature; PV, peak viscosity; RDS, rapidly

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digestible starch; RS, resistant starch; SB, setback viscosity; SDS, slowly digestible

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starch; To, onset temperature of gelatinization; Tp, peak temperature of gelatinization;

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∆H, enthalpy of gelatinization; SP, swelling power; HD, hardness; ADH, adhesiveness

chromatography;

HPSEC,

HPAEC, high-performance

high-performance

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ACS Paragon Plus Environment

size

exclusion


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ABSTRACT: We determined the relationships among the structural properties, in

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vitro digestibility and genetic factors in starches of 14 rice cultivars. Weight-based

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chain-length distributions in amylopectin ranged from 18.07% to 24.71% (fa, DP

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6-12), 45.01% to 55.67% (fb1, DP 13-24), 12.72% to 14.05% (fb2, DP 25-36) and

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10.80 to 20.72% (fb3, DP > 36), respectively. The contents of rapidly digestible starch

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(RDS), slowly digestible starch (SDS) and resistant starch (RS) ranged from 78.5% to

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87.5%, 1.2% to 6.0% and 10.1% to 18.0%, respectively. AAC was negatively

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correlated with RDS content, but positively correlated with RS content in rice starch.

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The proportion of short chains in amylopectin was positively correlated with RDS.

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Starch synthase IIa (SSIIa) gene controls the degree of crystallinity, the amount of fa

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chains of amylopectin. SSIIIa gene controls the amount of fb1 chains. Wx gene

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controls the amount of fraction I (FrI) and FrIIa fractionated by gel permeation

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chromatography (GPC), RDS, and RS. Starch debranching enzyme isoamylase II

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(ISA2) gene also controls the RDS, which may suggest that RDS was also affected by

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amylopectin structure although no correlation between them was found. This study

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indicated that genetics (i.e. starch biosynthesis related genes) controls structural

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properties of starch, and both amylose content and amylopectin fine structure

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determined functional properties of rice starch (i.e. the digestion), each in a different

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way. Understanding the ‘genetics-structure-function’ relationships in rice starches will

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assist plant breeders and food processors to develop new rice varieties and functional

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foods.

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KEYWORDS: rice starch, digestibility, genetics, starch structure, functionality

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INTRODUCTION

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As a staple food crop for half of the global population, rice has been consumed

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by humans for at least 5000 years. The digestive properties of starch have important

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potential implications for human health considering that starch is a primary dietary

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component. Starch can be categorized into rapidly digestible starch (RDS), slowly

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digestible starch (SDS) and resistant starch (RS).1 RDS is associated with elevated

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plasma glucose and insulin; consequently, it is linked with diabetes, coronary heart

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disease and the ageing process.2 SDS results in a slow increase of postprandial blood

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glucose levels and sustained blood glucose levels over time compared to RDS, which

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is advantageous for physical and mental performance, satiety and diabetes

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management.3 Therefore, SDS is the most desirable starch type from a nutritional

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point of view.2 RS has beneficial physiological effects through the production of short

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chain fatty acids during its fermentation in the large intestine.4 Possible relationships among starch structure and digestive properties have been

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documented.5-8

66

well

When

amylopectin

structure

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high-performance size exclusion chromatography (HPSEC), RDS content was

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negatively correlated with the amount of long and intermediate chains with DP >13,

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but positively correlated with short chains (DP 37) was negatively correlated with

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RDS content and positively correlated RS content,9 which was in agreement with the

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results from the HPSEC.7 Furthermore, apparent amylose content (AAC) was

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positively correlated with RS and SDS,5, 6 but negatively correlated with RDS. 5

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Based on the above investigation, the digestive properties of starch were

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influenced by both amylose content and amylopectin fine structure. However, the

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concerted interplay or contributions of amylose and amylopectin fine structures are

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not completely understood.

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Genetic investigations into the mechanisms underlying the formation of SDS and

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RS content in rice starches are scarce. Some starch mutants such as amylose extender,

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in which the starch branching enzyme IIb (BEIIb) gene is defective, displayed high

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RS.10 For a japonica high RS mutant “Jiangtangdao 1” with RS of 11.67%, BEIIb was

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found to be the causal gene for the elevated RS content.11 For another high amylose

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mutant in japonica background (Goami 2), BEIIb was not the causal gene,12 whereas

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the genetic controls for some other high RS mutants in indica background are still

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unknown.13 Low glycemic index (GI) food is able to assist in the management or

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prevention of type II diabetes, a major chronic disease in developing countries where

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rice is the staple food. In a study of 235 varieties, the Waxy gene was the major gene

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associated with GI.14 However, genetic control of the naturally occurring variations in


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SDS and RS contents in rice starch has not been reported.

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To deepen understanding of the “genetics – structure – functionality”

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relationships of rice starch, investigation of diverse rice starch samples with different

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amylose content and similar amylopectin fine structure or similar amylose content

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(for example, waxy rice) and different amylopectin fine structure combinations is

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required. Previously, we examined fourteen rice cultivars varying in the combination

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of AAC and gelatinization temperatures for their physicochemical and functional

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properties.15 In this study, the same series of rice starch samples were analyzed for

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starch structure and digestibility. The objectives were: i) to determine the contribution

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of amylose content and amylopectin fine structure to the in vitro digestion properties

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of the selected rice starches; ii) to delve into the genetic contribution of the starch

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synthesis related genes (SSRGs) to the starch structure and functional properties.

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MATERIALS AND METHODS

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Materials. Fourteen rice cultivars were selected in current study and included three

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waxy (Youzaonuo, Lishuinuo and Xiangnuo) and eleven normal (Zhefu504, Ce 482,

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93-11,Xiushui 11, Zhonghua11,Lemont, IR64, Jiayu293, Zhenshan B, Zaiyeqing 8

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and LongB) types as described in a previous report (Table 1).15 Sample preparation

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and starch isolation procedures were previously described.

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In vitro starch digestibility. Samples of the starch fraction (550 mg, dry weight basis)

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were weighed in a screw-capped tube and heated in 10 mL distilled water for 20 min



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with frequent mixing. Following this, 15 glass beads (0.5 mm diameter), guar gum

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(50 mg) and 10 mL of sodium acetate buffer (0.25 M, pH 5.2) were added to each

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tube. The tubes were then incubated with pancreatic α-amylase (50 units) and

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amyloglucosidase (35 units) at 37 °C in a shaking water bath. After 20 and 120 min of

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incubation, the hydrolysates (0.5 mL) were transferred to 20 mL of 80% ethanol to

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stop the reaction. The amount of released glucose was determined using a glucose

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oxidase assay kit (Megazyme, Bray, Co. Wicklow, Ireland). The percentages of

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glucose after 20 (SD20) and 120 (SD120) min incubations were calculated using a

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glucose standard curve to determine the percent starch digested. The contents of

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rapidly digestible starch (RDS), slowly digestible starch (SDS) and resistant starch

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(RS) in rice starch samples were determined according to a previous report.1

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2.3 Gel-permeation chromatography (GPC). Rice starch (~3.5 mg) was dissolved

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in 100 µL of dimethyl sulfoxide (DMSO) with constant stirring overnight. Hot MilliQ

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water (800 µL) and 100 µL of 0.1 M NaAc buffer (pH 4.5) were added to the

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starch-DMSO solution. Isoamylase (1 µL, from Pseudomonas sp., 1000 U/mL, EC

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3.2.1.68, Megazyme, Bray, Co. Wicklow, Ireland) and 1 µL of pullulanase (from

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Klebsiella planticola., 700 U/mL, EC 3.2.1.41, Megazyme, Bray, Co. Wicklow,

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Ireland) were added to start the reaction, which was conducted overnight with slow

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and constant stirring at room temperature. A NaOH solution (100 µL 5 M) was added

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to the debranched starch sample, centrifuged, and then analysed by gel-permeation

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chromatography (GPC) on a column (1 × 90 cm) of Sepharose CL 6B (Pharmacia,


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Uppsala, Sweden) with 0.5 M NaOH as an eluent at a rate of 0.5 mL/min. The column

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was calibrated by using dextrans with known molecular weights. The carbohydrate

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content of the collected fractions (0.5 mL) was determined with the phenol–sulfuric

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acid reagent.16

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Amylopectin

fractionation

and

chain-length

distribution

determination.

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Amylopectin fractionation followed previous methods based on butanol-alcohol

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precipitation.17,18 Purified amylopectin (9 mg) was dissolved in 450 µL 100% DMSO

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with constant stirring overnight. The solution was then diluted with 2250 µL of MilliQ

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water and 300 µL 0.1M sodium acetate buffer (pH 4.5), 1 µL of isoamylase (from

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Pseudomonas sp., 1000 U/mL, EC 3.2.1.68, Megazyme, Bray, Co. Wicklow, Ireland)

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and 1 µL of pullulanase (from Klebsiella planticola, 700 U/mL, EC 3.2.1.41,

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Megazyme, Bray, Co. Wicklow, Ireland) were added. The debranching reaction was

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conducted at room temperature with constant stirring overnight and was terminated by

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heating. The sample was centrifuged and filtered (pore size 0.45 µm) before injection

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into the HPLC system.

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The chain length distributions of debranched samples were analysed with a method

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modified from a previous report using a high-performance anion-exchange

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chromatography (HPAEC) system (Dionex ICS-5000+, Sunnyvale, CA) coupled with

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a BioLC gradient pump and a pulsed amperometric detector (PAD).18 The PAD signal

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was recorded by Chromeleon software (Version 6.8) and corrected to carbohydrate

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content.19 Prior to loading the sample, the column (250 mm × 4 mm, Carbo-Pac



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PA-100 with a guard column) was eluted at a rate of 1 mL/min with 150 mM NaOH

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for 20 min, then with a mixture of 150 mM NaOH (eluent A, 93%) and 150 mM

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NaOH containing 1 M NaOAc (eluent B, 7%) for 20 min. The elution gradient with a

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rate of 1 mL/min, was as follows: from 0 to 1.3 min, 93% eluent A; from 1.3 to 10

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min, eluent A changed from 93 to 82% linearly; from 10 to 19 min, from 82 to 78%;

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from 19 to 111 min, from 78 to 50%; from 111 to 113 min, from 50 to 93% (after

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which it returned to the starting mixture). The sample was injected during the initial

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1.2–1.3 min.

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Physicochemical properties. Parameters of the physicochemical properties used in

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the current study were described in our previous report.15

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Association test. Since all the 14 accessions were selected from our association

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panel,20-22 the genotypes of all the SSRGs (Supplementary Table 1) as well as other

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markers (i.e. microsatellites) were used in this study, to test the association among

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SSRGs, starch structure and functionality (digestive properties). Due to small amount

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of samples, the analysis of variance (ANOVA) and the kinship coefficients (K)

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models were performed with TASSEL Version 2.1 software. The P-value determining

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whether a marker is associated with a trait was set at P < 0.05.

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Statistical analysis. All analyses were determined in triplicate. Statistical analysis

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was conducted using SigmaPlot 9.01 integrated with SigmaStat 3.11 (Systat Software,


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CA). The results were subjected to one way analysis of variance (ANOVA) and

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Tukey's test to determine significant differences.

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RESULTS AND DISCUSSION

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Molecular structure characterization of rice starch. The debranched total starch

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samples were analyzed by gel-permeation chromatography (GPC) with Sepharose CL

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6B to determine the molecular size distribution profiles. The typical profiles from

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selected starch samples are shown in Fig. 1.Three major peaks were identified: the

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fraction from the first peak area (FrI) is amylose, while the fractions of the second and

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third peak areas (FrIIb and FrIIa) are long and short unit chains of amylopectin,

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respectively (Fig. 1). As expected, percentage of FrI from waxy rice starch was low

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(0.2-1.5%), while non-waxy rice starch had 16.7-25.6%. The content of FrI followed

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the general trend of AAC as determined by the iodine reagent method (Table 1). Since

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the amylose content obtained from Sepharose CL 2B (before debranching) and 6B

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(debranched) differ,23 caution should be taken when comparing the AAC obtained

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from the different methods.

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Purified amylopectin debranched by enzymes was employed to determine the

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chain-length profile. The parameters of chain-length distribution and average

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chain-length of amylopectin are summarized in Table 2, and a comparison of

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amylopectin chain-length distribution profiles from selected rice starch samples is

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presented in Fig. 2. The weight-based chain-length distribution of debranched

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amylopectin was grouped into four fractions: fa (DP 6-12), fb1 (DP 13-24), fb2 (DP



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25-36) and fb3 (DP > 36), according to the division suggested by a previous report.24

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The percentages of fa, fb1, fb2 and fb3 were 18.07%-24.71%, 45.01%-55.67%,

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12.72%-14.05% and 10.80%-20.72%, respectively. The average chain-length in rice

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amylopectin was DP 16.34-17.76, which was in agreement with a previous report on

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waxy rice;25 however, these values were lower than a recent report describing the

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average chain-length of six waxy rice starches, which ranged from DP 21.9 to 24.3.9

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In another study, the average chain-length of four waxy rice starches was from DP

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20.0 to 20.7.26 The results showed that amylopectin chain-length profiles from three

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waxy and 11 normal types was similar, which indicated that the amylopectin fine

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structure was independent of the amylose content. This result challenged a conclusion

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made by a previous report that amylose content was significantly correlated with

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amylopectin average chain-length and the ratio of long chains to short chains.27

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Amylopectin chains in high-amylose and intermediate-amylose rice were shown

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to be longer than in waxy and low-amylose rice when four rice cultivars were

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compared;7 however, when more cultivars were included as in this study, the trend

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was not clear (Table 2). Generally, waxy and low-amylose (BP005) rice starches had

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a higher degree of crystallinity than normal types. However, waxy type BP597 had a

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much lower degree of crystallinity. Notably, fb1 (DP 13-24) chains in BP597 were

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lower and fb3 (DP >36) were higher than the other two waxy types (Table 2),

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indicating that fb1 chains contributed to the formation of crystallinity while fb3

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hindered this formation. Normal type BP003 had a higher degree of crystallinity than

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other normal types, and the highest proportion of fb1 chains and lowest proportion of


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fb3 chains was observed in BP003 (Table 2), which further supported the roles of fb1

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and fb3 chains in starch crystallinity. It has also been suggested that fb1 chains form

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double helices that span the entire length of crystalline regions.28

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In vitro digestibility of rice starch. The in vitro methodology for determining

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nutritional starch fractions was developed by Englyst and coworkers1 and describes

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the likely rate and extent of glucose release from foods in the small intestine; it has

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been verified to match the mean proportion of starch recovered in ileostomy studies.29,

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30

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fate of the carbohydrate when it is consumed.31

These nutritional fractions determine the bioavailability and likely physiological

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The nutritional fractions of rice starch, including rapidly digestible starch (RDS),

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slowly digestible starch (SDS) and resistant starch (RS), are presented in Table 3. The

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RDS content of rice starch ranged from 78.5 to 87.5%. The lowest and highest RDS

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contents were observed in BP028 and BP011, respectively. Waxy rice starch types had

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relatively higher RDS content than normal types. SDS content from rice starch

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samples ranged from 1.2 to 6.0%, with the lowest content observed in BP011 and the

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highest in BP005. RS content varied from 10.1% (BP005) to 18.0% (BP578) (Table

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3). It has been proposed that combining the fractions of SDS and RS was useful for

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comparison of starch types,32 and normal rice starch showed relatively higher

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fractions of SDS and RS than waxy types, this phenomenon was also observed in

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current study. Rice starch digestion properties were previously shown to be affected

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by amylopectin fine structure,5 starch fine molecular structure and crystalline



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structure.9 RS has been correlated positively with amylose content in rice starch, while

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SDS decreased as amylose content increased.7 RDS content of rice starches decreased

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with increasing amylose content,6 and waxy starches showed greater enzyme digestion

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than normal types.33 Higher RDS content in waxy rice starch was attributed to the

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lower proportion of long chains in amylopectin (DP ≥ 37), the molecular weight in

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molecular structure, the onset temperature of gelatinization and crystallinity, and the

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higher proportion of short A (DP 6-12) chains.9

257 258

Relating starch structure to functional and digestion properties. Starch

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physicochemical and functional parameters including AAC, starch crystallinity,

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pasting and thermal properties were adapted from our previous report.15 The fraction

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of short chains (DP 6-12), fa, was negatively correlated with the degree of

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crystallinity (r = −0.67, p < 0.01) (Table 4). AAC was previously shown to be

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negatively correlated with the degree of crystallinity (r = −0.62, p < 0.001).15 However,

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a higher proportion of short chains (DP10-13) resulted in a higher degree of

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crystallinity and the degree of starch crystallinity decreased with increasing amylose

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content in a previous report.34 Waxy types BP011 and BP601, and normal types

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BP028 and BP578 exhibited similar amylopectin chain-length profiles regardless of

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amylose content (Table 2). The waxy types had a higher degree of crystallinity than

269

the normal. Waxy type BP597 had lower proportion of fb1 chains and a relatively

270

higher proportion of fb3 chains than the other two waxy types, these differences in

271

amylopectin structure contributed to a lower degree of crystallinity in BP597. Based


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on the above, the starch crystallinity was not affected by amylose content or

273

amylopectin fine structure alone, but by the interplay of amylose and amylopectin fine

274

structure.

275

FrI consisted of debranched amylose and represented the amylose content, so it

276

is not surprising that AAC showed a significant positive correlation with FrI (r = 0.97,

277

p < 0.001) (Table 4). Breakdown viscosity (BD) was positively correlated with the

278

amout of short chains (fa), but negatively correlated with the amount of fb1 chains.

279

Previous study also indicated that rice starches with higher proportions of fb1 chains

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seemingly had lower BD, whereas with higher proportions of fa chains had larger

281

BD.35 These observations were generally consistent with our current results. No

282

significant correlation between BD and FrI was observed, however, FrI was positively

283

correlated with CPV (r = 0.78, p < 0.001) and SB (r = 0.82, p < 0.001) (Table 4).

284

When the starch paste was cooled, aggregation of leached amylose resulted in a strong

285

gel due to the formation of hydrogen bonds from free amylose, which explained the

286

current observed results, and similar observations were reported previously in rice

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starches with different amylose contents.6 Gelatinization parameters, including To, Tp

288

and ∆H, had negative correlations with the proportion of short fa (DP 6-12) chains, fa

289

chains were also negatively correlated with RVA pasting temperature (r = −0.84, p
36) cd

CLap(DP)

BP011 BP597 BP601

18.07 22.26b 19.58c

52.94 47.11e 51.38c

14.05 13.45ab 13.12bc

14.93 17.18b 15.92bc

17.61a 17.01bc 17.38ab

BP005 BP025 BP033 BP050 BP605

18.18e 22.48b 24.71a 23.03ab 22.28b

51.59bc 49.84cd 48.51d 48.73d 49.34d

13.00bc 13.82a 13.26bc 13.39bc 13.59ab

17.23b 13.86e 13.52e 14.86cd 14.79cd

17.76a 16.63cd 16.34d 16.60cd 16.73cd

BP015 BP047

20.06cd 21.55b

53.52b 45.01f

13.21bc 12.72c

13.21e 20.72a

17.06bc 17.54a

BP003 BP028 BP578 BP628

20.46cd 18.98de 19.44de 20.11cd

55.67a 52.37bc 52.04bc 51.17c

13.07bc 13.46ab 13.11bc 13.00bc

10.80f 15.20bcd 15.40bcd 15.72bcd

16.71cd 17.44ab 17.31ab 17.21ab

Values in the same column with different superscripts are significantly different (p < 0.05).



555 556 557

Table 3 Digestion properties of starches from selected fourteen rice cultivars 558 RS (%) 559 BP011 87.5 ± 1.4 88.7 ± 1.2 87.5a 1.2c 11.3e 560 BP597 80.0 ± 0.9 83.8 ± 1.4 80.0ef 3.8b 16.2bc 561 BP601 85.8 ± 0.6 89.4 ± 1.4 85.8ab 3.7b 10.6e 562 563 BP005 84.0 ± 1.0 90.0 ± 0.9 84.0bc 6.0a 10.1e 564 2.9bc 16.5abc BP025 80.6 ± 0.9 83.5 ± 1.0 80.6e 565 BP033 81.5 ± 1.6 84.8 ± 1.0 81.5d 3.4b 15.2c 566 3.0bc 14.0d BP050 83.1 ± 1.0 86.0 ± 1.4 83.1c 567 BP605 85.5 ± 0.8 89.4 ± 0.8 85.5b 3.9b 10.6e 568 569 BP015 82.8 ± 1.2 86.1 ± 1.1 82.8cd 3.3b 13.9d 570 BP047 81.2 ± 1.0 84.6 ± 1.4 81.2de 3.4b 15.4c 571 572 BP003 79.5 ± 0.8 82.8 ± 0.5 79.5e 3.4b 17.2ab 573 BP028 78.5 ± 1.1 82.5 ± 0.6 78.5f 4.1ab 17.5ab 574 BP578 80.6 ± 1.0 82.0 ± 0.6 80.6e 1.4c 18.0a 575 BP628 80.4 ± 1.1 83.3 ± 0.6 80.4ef 2.9bc 16.7abc 576 Values in the same column with different superscripts are significantly different (p < 0.05). Code

577 578 579 580 581 582 583 584 585 586 587 588 589 590 591 592

SD20 (%)

SD120 (%)

RDS (%)

SDS (%)


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593 594 595 596

Table 4 Correlations between starch structural parameters and functional properties. AAC fa fb1 fb2 fb3 CLap FrI FrIIa FrIIb

597 598 599 600 601 602 603 604 605 606 607 608 609 610 611 612 613 614 615 616 617 618 619 620 621 622 623 624 625 626 627

0.02 0.20 –0.40 –0.21 –0.15 0.97*** –0.94*** –0.85***

Crystallinity –0.67 0.48 0.16 0.03 0.55* –0.46 0.51 0.31

**

BD

CPV *

0.58 –0.54* 0.43 0.09 –0.43 –0.19 0.15 0.21

–0.20 0.35 –0.54* –0.16 0.07 0.78*** –0.76** –0.69**

SB –0.41 0.55* –0.50 –0.23 0.20 0.82*** –0.59* –0.63*

* ** ***

PTemp

To ***

–0.84 0.63* –0.21 –0.01 0.69** –0.02 0.05 0.03

, , Correlations are significant at P < 0.05,P < 0.01, P < 0.001, respectively.

29


Tp **

–0.77 0.45 0.02 0.12 0.72** –0.23 0.22 0.20

∆H **

–0.77 0.38 –0.03 0.21 0.77** –0.30 0.27 0.30

–0.56* 0.04 0.11 0.43 0.73** –0.42 0.32 0.52


628 629 630 631

Table 5 Correlations between rice starch digestion properties and structural/functional parameters. RDS FrI FrIIa FrIIb fa fb1 fb2 fb3 CLap Crystallinity AAC SP PV BD CPV SB To Tp ∆H HD ADH

632 633 634 635 636 637 638 639 640 641 642 643 644 645 646 647 648 649 650

* ** ***

, ,

*

–0.56 0.56* 0.46 –0.23 0.05 0.34 0.08 0.23 0.51 –0.62* 0.78** –0.46 –0.04 –0.66** –0.38 0.28 0.36 0.19 –0.75** 0.63*

SDS

RS

0.06 –0.12 0.04 0.01 –0.13 –0.36 0.21 0.10 –0.09 –0.03 –0.13 0.40 0.09 0.13 –0.08 –0.11 –0.06 –0.08 –0.17 0.19

0.51 –0.49 –0.46 0.21 0.01 –0.17 –0.17 –0.26 –0.44 0.60* –0.67* 0.26 –0.01 0.58* 0.40 –0.22 –0.31 –0.15 0.76** –0.66*

Correlations are significant at P < 0.05, P < 0.01, P < 0.001, respectively.

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651 652 653

Table 6 Putative SSRG markers in relation to starch crystallinity, structure and functionality revealed by ANOVA and Kinship (K) models Trait

Locus

Crystallinity SSIIa CLap SSIIa SSIVb Fa SSIIa Fb1 SSIIIa Fb2 AGPL1 Wx FrI AGPL1 Wx FrII AGPL1 Wx FrIIa Wx AGPL1 FrIIb AGPL1 Wx RDS Wx ISA2 RS Wx

Chr

6 6 5 6 8 5 6 5 6 5 6 6 5 5 6 6 5 6

Anova model P 0.0014 0.0051 0.0482 0.0013 0.0263 0.0217

K model R2 0.5867 0.493 0.3099 0.5911 0.3484 0.3667

0.0048 0.0093 0.0048 0.0093 0.0055 0.0064 0.0230

0.4977 0.4434 0.4977 0.4434 0.4879 0.4755 0.3613

0.0300 0.0374 0.0179

0.3353 0.3374 0.385

654 655 656 657 658 659 660 661 662 663 664 31


P 0.0014 0.0051 0.0483 0.0013 0.0263

R2 0.5853 0.4921 0.3092 0.5893 0.3477

0.0508 0.0048 0.0060 0.0048 0.0060 0.0054 0.0038 0.0231 0.0325 0.0301 0.0372 0.0179

0.2816 0.4944 0.2747 0.4944 0.2747 0.4839 0.193 0.3604 0.211 0.3294 0.2917 0.3821


665 666 667 668 669

Fig. 1.

32


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670 671 672 673 674

675 676 677 678 679

Fig.2.

680 681 682 683 684 685 686 687 688 689 690 33



691

Graphic for TOC

692 693

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Relationships among Genetic, Structural, and Functional Properties of

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