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Turkish Journal of Agriculture and Forestry Turk J Agric For(2018) 42: 248-261© TÜBİTAKdoi:10.3906/tar-1801-95

Isoflavone profile diversity in Korean wild soybeans (Glycine soja Sieb. & Zucc.)

Chigen TSUKAMOTO1, Muhammad Amjad NAWAZ2

, Ayaka KUROSAKA2, Bao LE2

, Jeong Dong LEE3,

Eunho SON4, Seung Hwan YANG2

, Cemal KURT5, Faheem Shehzad BALOCH6

, Gyuhwa CHUNG2,*1Faculty of Agriculture, Iwate University, Iwate, Japan

2Department of Biotechnology, Chonnam National University, Chonnam, Republic of Korea 3Division of Plant Biosciences, Kyungpook National University, Daegu, Republic of Korea

4National Agrobiodiversity Center, National Institute of Agricultural Sciences,Rural Development Administration, Jeonju, Republic of Korea

5Department of Field Crops, Faculty of Agriculture, Çukurova University, Adana, Turkey6Department of Field Crops, Faculty of Agriculture and Natural Sciences, Abant İzzet Baysal University, Bolu, Turkey

* Correspondence: chung@chonnam.ac.kr

1. IntroductionSoybean [Glycine max (L.) Merr.] seeds are one of the major affordable sources of nutrients in the diets of animals and humans. They are rich in protein, oil, dietary fiber, lipids, secondary metabolites, essential minerals, and physiologically active metabolites, such as isoflavones, tocopherols, and saponins (Sugano, 2005). Isoflavones are a subclass of polyphenols ubiquitous in more than 300 types of plants (Klejdus et al., 2005). Major sources of isoflavones are plant species belonging to the family Fabaceae, such as lupines, kudzu, faba beans, and soybeans, as well as many other legume plants (Wang and Murphy, 1994a; Barnes et al., 2010; Prasad et al., 2010). Isoflavones have been reported to control and prevent the incidence of cancer, decrease cardiovascular problems, and reduce menopausal symptoms (Nawaz et al., 2018).

The use of soy-based disease-preventing functional foods has escalated over the past two decades (Kuiper et al., 1997; Cvejic et al., 2011). Genistin and daidzin have

antioxidant functions, as well as acting as potent scavengers (Foti et al., 2005; Rufer and Kulling, 2006). Soy-isoflavones have antitumor and antiinflammatory functions. Protein tyrosine kinase inhibitors in soy-isoflavones play a role in improving cardiovascular function and inhibition of growth and proliferation of gastric cancer cells (Piontek et al., 1993; Yu et al., 2012). The role of soy-isoflavones in activating endothelial nitric oxide synthase, elevating prostacyclin levels, inhibiting cell proliferation, relaxing vessels, and reducing plaque has also been studied (Cano et al., 2010; Ko, 2014). In addition, isoflavones aid in the promotion of nodulation by rhizobia, microorganism cluster changes around roots, and biotic stress resistance (Zeng et al., 2009). Until now, soy-isoflavone contents have been published focusing on total amount and factors influencing quantity (Wang and Murphy, 1994a, 1994b; Lee et al., 2003; Genovese et al., 2005, 2006; Lee et al., 2007; Devi et al., 2009). It is now established that the soy-isoflavone content in seeds of the same genotype is greatly

Abstract: Isoflavones prevent the incidence of cancer, reduce cardiovascular problems, and decrease menopausal symptoms. In soybean (Glycine soja Sieb. & Zucc.) seeds, the content is greatly affected by growing conditions. The variations in soy-isoflavone content were investigated by growing 346 Korean wild soybean accessions from natural habitats under the same agroclimatic conditions. High-performance liquid chromatography was used to measure the profiles of six soy-isoflavones, i.e. glycosides (daidzin, glycitin, genistin) and malonyl glycosides (malonyl-daidzin, malonyl-glycitin, and malonyl-genistin). The soy-isoflavone content of the wild soybeans ranged from 0.5 to 5.51 mg/g with an average of 2.78 mg/g. The highest mean total soy-isoflavone was genistin (1.393 mg/g), followed by daidzin (0.939 mg/g) and glycitin (0.250 mg/g). Korean wild soybean seeds examined in this study were rich in malonyl-genistin. An increasing trend for total soy-isoflavone content was observed from the north to the southeast in the geographical distribution sites, which signifies the effect of genotype and collection site. The information generated in this report will greatly aid soybean breeding stratagems aimed at improving soy-isoflavone concentration in soybean seeds.

Key words: Daidzin, isoflavone, Glycine soja, genistin, glycitin, Korean wild soybeans

Received: 20.01.2018 Accepted/Published Online: 28.03.2018 Final Version: 07.00.2018

Research Article

This work is licensed under a Creative Commons Attribution 4.0 International License.

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affected by environmental conditions (different dates of planting, years of growing, and locations) that occur during seed development (Tsukamoto et al., 1995; Hoeck et al., 2000). The compositional changes are less distinctive (Wang and Murphy, 1994a).

The reducing effect of increased temperature, carbon dioxide, and water stress on soy-isoflavone content has been reported (Caldwell et al., 2005). To breed soybean cultivars with higher soy-isoflavone content, identification of the genes or quantitative trait loci (QTLs) controlling soy-isoflavone content has top priority for marker-assisted selection. Thus far, many genomic regions (A1, B1, B2, C2, D1a_Q, F, G, H, J, K, M, and N) have been associated with individuals along with total soy-isoflavone content in soybeans (Primomo et al., 2005; Kassem et al., 2006; Zeng et al., 2009; Gutierrez-Gonzalez et al., 2010; Zhao et al., 2015). Recombinant inbred lines for soy-isoflavone content were previously used for identification of QTLs, which was supported by next-generation sequencing data and molecular markers. However, genes controlling soy-isoflavone content and individual types are still undetermined. To identify genes regulating soy-isoflavone biosynthesis, the first step is to investigate genetic variation (excluding environmental effects) and to determine candidate accessions having higher or lower soy-isoflavone content in the seeds. For this, exploration of soy-isoflavone content of wild soybean (Glycine soja Sieb. & Zucc.) is a rational choice because of wide genetic diversity (Baloch et al., 2010; Wang et al., 2016; Nawaz et al., 2017). For this type of screening, wild soybeans of the Korean peninsula grown with the same environmental conditions, locations, and years were the specimens used to determine genetic diversity. The variation revealed in this study will aid in identification of genes and/or gene combinations to breed soybean cultivars with higher soy-isoflavone contents. The possibility of identifying natural mutants through large-scale germplasm screening may also be fruitful (Tsukamoto et al., 1995; Zhang et al., 2012).

Searching for high soy-isoflavone content-containing genotypes is a never-ending process. Previous studies have suggested that total soy-isoflavone content varied among different countries, such as the United States, China, Korea, Ecuador, and Brazil. Korean wild soybeans contained the maximum amounts of isoflavone (Wang and Murphy, 1994; Lee et al., 2003; Genovese et al., 2005, 2006; Lee et al., 2007; Devi et al., 2009). However, comprehensive reports are not available on the geographical distribution of soy-isoflavone in the Korean peninsula. The center of wild soybean diversity (China, Korean peninsula, and to some extent Japan) may represent abundant soy-isoflavone content and component variations. This valuable information is missing and should be generated to improve breeding programs to develop soybean cultivars with higher soy-isoflavone

contents. In this study, we focused on determining soy-isoflavone content and composition [daidzin, glycitin, genistin, malonyl-daidzin (m-daidzin), malonyl-glycitin (m-glycitin), and malonyl-genistin (m-genistin)] in Korean wild soybean germplasm originating from all geographic locations in the Korean territory.

2. Materials and methods2.1. Plant materialThree hundred and forty-six wild soybean accessions belonging to South Korean wild soybean habitats were selected from Chung’s Wild Legume Germplasm Collection (CWLGC, Chonnam National University, Yeosu, South Korea). Care was taken during the selection of the available accessions that the maximum number of accessions were selected to represent all major and minor wild soybean habitats (Figure 1; Table 1).

Accessions were grown in Jinju, South Korea (35.1800°N, 128.1076°E), during the 2016 growing season under natural climatic conditions with a plant-to-plant distance of 2 m and row-to-row distance of 2 m according to a completely randomized design with four replications. The meteorological data (humidity, temperature, and rainfall) of all Korean provinces and cities of 2017 and past years can be accessed through the website of the Korean Meteorological Administration (www.weather.go.kr). After the complete maturation of seeds, pods of each accession were harvested and threshed separately and processed for sample preparation. 2.2. Sample preparationHarvested dried seeds (10 seeds of each accession) were manually ground to powder with a hammer. A 10-fold volume of aqueous methanol (80%) was added to the soybean seed powder, which was pulverized twice with a multibead shaker for 15 s and extracted for 1 h at room temperature. The extract was then centrifuged at 12,000 rpm for 5 min and the supernatant was removed for use in further analysis. 2.3. HPLC analysis of soy-isoflavone content and compositionHigh-performance liquid chromatography (HPLC) analysis was conducted using an HPLC-photodiode array detector and analyzer system (LC9A, Shimadzu Corporation, Kyoto, Japan) with a Develosil C-30 UG-3 (I.D. 2.0 × 150 mm) column following an improved and modified method of Sun et al. (2011). The solvent flow rate was 0.15 mL/min with an injection volume of 20 µL. Wavelength of the UV detector was adjusted to 254 nm. The mobile phase gradient program was employed for the analysis as follows: 0 min, 85% A/15% B; 0–30 min, 55% A/45% B; washing 0% A/100% B, 30–35 min; recycling to the initial condition 85% A/15% B, 15 min; total analysis time: 60 min. Solvent A consisted of acetonitrile containing

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0.1% (v/v) formic acid and solvent B was 0.1% formic acid solution.

Purified daidzin was purchased from Nakalai Tesque (Japan) and dissolved in 70% aqueous ethanol (v/v) containing 0.1% acetic acid (v/v). Daidzin solution (mg/mL) was used for preparing the calibration curve of crude soy-isoflavone solution containing three soy-isoflavones, daidzin, glycitin, and genistin. The crude isoflavone solution was used as a standard in HPLC analysis and determination. Analysis of the crude isoflavone solution was conducted once in a repetitive manner before the analysis of 25 samples.2.4. Statistical analysisQuantification data from HPLC for total soy-isoflavone content (mg/g; milligrams per gram of wild soybean seed) and composition of each accession were used for statistical

analysis in SPSS (SPSS Inc., Chicago, IL, USA). The data were analyzed by using analysis of variance (ANOVA) and t-tests, with a significance level of P < 0.05. Frequency distribution of total genistin, daidzin, and glycitin and total soy-isoflavones was calculated in Microsoft Excel. We performed principal component analysis (PCA) for soy-isoflavone content and composition (i.e. six individual soy-isoflavones) in the studied Korean wild soybean accessions. To explore geographical trends (spatial analysis), Image Analyst, Spatial Analyst, and Patch Analyst extensions implemented in ArcGis 9.3 software were used (http://www.cnfer.on.ca/SEP/patchanalyst/whats_new.html).

3. ResultsThe six soy-isoflavones were successfully identified through applied isolation conditions and HPLC analysis

Figure 1. Geographical distribution of selected Korean wild soybean accessions.

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Table 1. Selected wild soybean accessions and their collection sites.

No. Accession Collection site No. Accession Collection site

1 CW13229 Gangwon Gangneung 174 CW15964 Gyeongbuk Yangcheon2 CW13274 Gangwon Samcheok 175 CW15985 Gyeongbuk Yangpong3 CW13766 Gangwon Yangu 176 CW15963 Gyeongbuk Yocheon4 CW15912 Gangwon Yangu 177 CW15889 Gyeongbuk Yocheon5 CW15958 Gangwon Yangu 178 CW16054 Gyeongbuk Yocheon6 CW15973 Gangwon Yangu 179 CW15988 Gyeongbuk Yocheon7 CW15052 Gangwon Yungwol 180 CW13285 Gyeongbuk Uljin8 CW15919 Gangwon Yungwol 181 CW15281 Gyeongbuk Oeseong9 CW15920 Gangwon Yungwol 182 CW15998 Gyeongbuk Oeseong10 CW16042 Gangwon Yungwol 183 CW15997 Gyeongbuk Oesong11 CW15951 Gangwon Wonseong 184 CW15278 Gyeongbuk Oeseong12 CW15948 Gangwon Wonseong 185 CW16007 Gyeongbuk Cheongdo13 CW15370 Gangwon Wonseong 186 CW15910 Gyeongbuk Cheongdo14 CW15947 Gangwon Wonseong 187 CW14672 Gyeongbuk Cheongdo15 CW15909 Gangwon Wonju 188 CW15911 Gyeongbuk Cheongdo16 CW15908 Gangwon Wonju 189 CW16050 Gyeongbuk Chilguk17 CW13714 Gangwon Wonju 190 CW16051 Gyeongbuk Chilguk18 CW16041 Gangwon Wonju 191 CW13319 Gyeongbuk Pohang19 CW15914 Gangwon Injae 192 CW16953 Gyeongbuk Pohang20 CW15913 Gangwon Injae 193 CW16950 Gyeongbuk Pohang21 CW15978 Gangwon Injae 194 CW16948 Gyeongbuk Pohang22 CW15976 Gangwon Injae 195 CW15849 Gwangju Namgu23 CW15977 Gangwon Injae 196 CW15843 Gwangju Namgu24 CW16046 Gangwon Jeongsan 197 CW12878 Jeonnam Gangchin25 CW15961 Gangwon Jeongsan 198 CW10846 Jeonnam Guhong26 CW15918 Gangwon Jeongsan 199 CW15855 Jeonnam Kukseong27 CW16044 Gangwon Jeongsan 200 CW17010 Jeonnam Gwanyang28 CW16047 Gangwon Jeongsan 201 CW17008 Jeonnam Gwanyang29 CW16045 Gangwon Jeongsan 202 CW12376 Jeonnam Gwangju30 CW15952 Gangwon Cheolwon 203 CW17032 Jeonnam Gwangju31 CW15953 Gangwon Cheolwon 204 CW17035 Jeonnam Gwangju32 CW15942 Gangwon Chunseong 205 CW17036 Jeonnam Gwangju33 CW15943 Gangwon Chunseong 206 CW11288 Jeonnam Gorae34 CW15892 Gangwon Chunseong 207 CW15840 Jeonnam Gorae35 CW15946 Gangwon Chunseong 208 CW12208 Jeonnam Muan36 CW15959 Gangwon Chunseong 209 CW15802 Jeonnam Muan37 CW13798 Gangwon Chuncheon 210 CW15799 Jeonnam Muan38 CW17038 Gangwon Chuncheon 211 CW15796 Jeonnam Muan39 CW17040 Gangwon Chuncheon 212 CW15838 Jeonnam Boseong40 CW17043 Gangwon Chuncheon 213 CW15870 Jeonnam Suncheon41 CW17064 Gangwon Chuncheon 214 CW15861 Jeonnam Suncheon42 CW17061 Gangwon Chuncheon 215 CW01012 Jeonnam Sinan

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Table 1. (Continued).

43 CW17053 Gangwon Chuncheon 216 CW12133 Jeonnam Sinan 44 CW17047 Gangwon Chuncheon 217 CW17145 Jeonnam Yeosu45 CW15916 Gangwon Pyeongchang 218 CW17068 Jeonnam Yeosu46 CW15981 Gangwon Pyeongchang 219 CW17151 Jeonnam Yeosu47 CW15917 Gangwon Pyeongchang 220 CW17146 Jeonnam Yeosu48 CW15902 Gangwon Pyeongchang 221 CW17152 Jeonnam Yeosu49 CW15980 Gangwon Pyeongchang 222 CW17147 Jeonnam Yeosu50 CW13938 Gangwon Pyeongchang 223 CW17149 Jeonnam Yeosu51 CW13818 Gangwon Hongcheon 224 CW13454 Jeonnam Yanggwang52 CW16009 Gangwon Hongcheon 225 CW15790 Jeonnam Yanggwang53 CW13749 Gangwon Howacheon 226 CW15788 Jeonnam Yanggwang54 CW16012 Gangwon Howacheon 227 CW15789 Jeonnam Yanggwang55 CW16035 Gangwon Howacheon 228 CW15883 Jeonnam Yanggwang56 CW16017 Gangwon Howacheon 229 CW15786 Jeonnam Yanggwang57 CW16039 Gangwon Howacheon 230 CW15783 Jeonnam Yanggwang58 CW16036 Gangwon Howacheon 231 CW12345 Jeonnam Yangam59 CW13848 Gangwon Huiseong 232 CW15866 Jeonnam Wondo60 CW15188 Gyeongi Goyang 233 CW15772 Jeonnam Changseong61 CW17031 Gyeongi Gwangmyon 234 CW15773 Jeonnam Changseong62 CW15095 Gyeongi Gwangju 235 CW14730 Jeonnam Changseong63 CW15090 Gyeongi Gwangju 236 CW15770 Jeonnam Changseong64 CW15109 Gyeongi Namyangchu 237 CW15771 Jeonnam Changseong65 CW13193 Gyeongi Seongnam 238 CW15856 Jeonnam Changheng66 CW14131 Gyeongi Ansan 239 CW15852 Jeonnam Changheng67 CW14911 Gyeongi Osan 240 CW15839 Jeonnam Changheng68 CW15859 Gyeongi Ilsan 241 CW15867 Jeonnam Changheng69 CW15857 Gyeongi Ilsan 242 CW11065 Jeonnam Jindu70 CW15878 Gyeongi Paju 243 CW15795 Jeonnam Hampyeong71 CW15172 Gyeongi Paju 244 CW15888 Jeonnam Hampyeong72 CW13385 Gyeongi Pyeongtek 245 CW15794 Jeonnam Hampyeong73 CW13367 Gyeongi Huwaseong 246 CW15781 Jeonnam Hampyeong74 CW17160 Gyeongnam Gojae 247 CW15875 Jeonnam Hampyeong75 CW17163 Gyeongnam Gojae 248 CW15778 Jeonnam Hampyeong76 CW17162 Gyeongnam Gojae 249 CW15776 Jeonnam Hampyeong77 CW17175 Gyeongnam Gojae 250 CW15774 Jeonnam Hampyeong78 CW13139 Gyeongnam Gojae 251 CW16726 Jeonnam Haenam79 CW15747 Gyeongnam Gachang 252 CW16727 Jeonnam Haenam80 CW15753 Gyeongnam Gachang 253 CW10917 Jeonnam Haenam81 CW15744 Gyeongnam Gachang 254 CW17141 Jeonnam Huasun82 CW11386 Gyeongnam Gosang 255 CW12521 Jeonnam Huasun 83 CW17228 Gyeongnam Namhae 256 CW12393 Jeonbuk Gochung84 CW17229 Gyeongnam Namhae 257 CW15769 Jeonbuk Gunsan

85 CW17234 Gyeongnam Namhae 258 CW15763 Jeonbuk Gimjae

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86 CW17235 Gyeongnam Namhae 259 CW15762 Jeonbuk Gimjae87 CW17230 Gyeongnam Namhae 260 CW15741 Jeonbuk Gimjae88 CW17232 Gyeongnam Namhae 261 CW12498 Jeonbuk Gimjae89 CW17233 Gyeongnam Namhae 262 CW14177 Jeonbuk Namwon90 CW13183 Gyeongnam Namhae 263 CW15820 Jeonbuk Nunsan91 CW13641 Gyeongnam Milyang 264 CW13440 Jeonbuk Buan92 CW10336 Gyeongnam Sacheong 265 CW15768 Jeonbuk Buan93 CW17259 Gyeongnam Sacheong 266 CW15767 Jeonbuk Buan94 CW17291 Gyeongnam Sacheong 267 CW12899 Jeonbuk Suncheong95 CW17260 Gyeongnam Sacheong 268 CW16922 Jeonbuk Wonju96 CW17249 Gyeongnam Sacheong 269 CW17292 Jeonbuk Wonju97 CW17207 Gyeongnam Sacheong 270 CW14201 Jeonbuk Wonju98 CW17199 Gyeongnam Sacheong 271 CW17293 Jeonbuk Wonju99 CW17202 Gyeongnam Sacheong 272 CW15864 Jeonbuk Iksan100 CW14688 Gyeongnam Sacheong 273 CW12511 Jeonbuk Iksan101 CW16185 Gyeongnam Yangsan 274 CW15863 Jeonbuk Iksan102 CW14713 Gyeongnam Oeleong 275 CW15200 Jeonbuk Changsu103 CW15743 Gyeongnam Jinju 276 CW16923 Jeonbuk Jeonju104 CW15755 Gyeongnam Jinju 277 CW15739 Jeonbuk Jeonju105 CW15758 Gyeongnam Jinju 278 CW15871 Jeonbuk Jeonju106 CW17192 Gyeongnam Jinju 279 CW15845 Jeonbuk Jeonju107 CW17216 Gyeongnam Jinju 280 CW15850 Jeonbuk Jeonju108 CW16343 Gyeongnam Changwon 281 CW15742 Jeonbuk Jeonju109 CW17153 Gyeongnam Tongyang 282 CW12450 Jeonbuk Cheongop110 CW17182 Gyeongnam Tongyang 283 CW15865 Jeonbuk Cheongop111 CW17224 Gyeongnam Hadong 284 CW15212 Jeonbuk Jinan112 CW17226 Gyeongnam Hadong 285 CW10118 Jeju113 CW17288 Gyeongnam Hadong 286 CW10115 Jeju114 CW17186 Gyeongnam Hadong 287 CW10110 Jeju115 CW17225 Gyeongnam Hadong 288 CW16532 South Jeju116 CW14869 Gyeongnam Haman 289 CW16521 North Jeju117 CW17254 Gyeongnam Hamyang 290 CW17278 Jeju Saguipo118 CW17253 Gyeongnam Hamyang 291 CW17279 Jeju Saguipo119 CW17255 Gyeongnam Hamyang 292 CW17280 Jeju Saguipo120 CW17243 Gyeongnam Hamyang 293 CW17284 Jeju Saguipo121 CW17241 Gyeongnam Hamyang 294 CW17283 Jeju Saguipo122 CW17238 Gyeongnam Hamyang 295 CW15929 Jeju Aewol123 CW17236 Gyeongnam Hamyang 296 CW17287 Jeju124 CW17247 Gyeongnam Hamyang 297 CW17274 Jeju 125 CW17248 Gyeongnam Hamyang 298 CW17276 Jeju126 CW17290 Gyeongnam Hamyang 299 CW17286 Jeju127 CW11977 Gyeongnam Hamyang 300 CW17285 Jeju128 CW17251 Gyeongnam Hamyang 301 CW17262 Jeju Chujah129 CW13348 Gyeongbuk Gyeongju 302 CW17265 Jeju Chujah

Table 1. (Continued).

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Table 1. (Continued).

130 CW16005 Gyeongbuk Goryang 303 CW17263 Jeju Chujah131 CW16006 Gyeongbuk Goryang 304 CW17268 Jeju Chujah132 CW16988 Gyeongbuk Ghumi 305 CW17269 Jeju Chujah133 CW16975 Gyeongbuk Ghumi 306 CW17271 Jeju Chujah134 CW16053 Gyeongbuk Ghumi 307 CW17272 Jeju Chujah135 CW16981 Gyeongbuk Ghumi 308 CW17273 Jeju Chujah136 CW16982 Gyeongbuk Ghumi 309 CW12636 Chungnam Gongju137 CW16275 Gyeongbuk Gunwui 310 CW14210 Chungnam Gimsan138 CW16000 Gyeongbuk Gunwui 311 CW15844 Chungnam Nunsan139 CW15999 Gyeongbuk Gunwui 312 CW14549 Chungnam Dangjin140 CW15890 Gyeongbuk Gimryong 313 CW16564 Chungnam Daejon141 CW16002 Gyeongbuk Gimryong 314 CW13411 Chungnam Boryong142 CW15922 Gyeongbuk Gimryong 315 CW12731 Chungnam Buyang143 CW15923 Gyeongbuk Gimryong 316 CW14526 Chungnam Seonsan144 CW16003 Gyeongbuk Gimryong 317 CW12709 Chungnam Sacheon145 CW16049 Gyeongbuk Dalseong 318 CW14568 Chungnam Asan146 CW16048 Gyeongbuk Dalseong 319 CW16298 Chungnam Yosan147 CW15937 Gyeongbuk Mongyeong 320 CW13400 Chungnam Yosan148 CW15927 Gyeongbuk Mongyeong 321 CW12586 Chungnam Cheonan149 CW15894 Gyeongbuk Mongyeong 322 CW14487 Chungnam Tehan150 CW15990 Gyeongbuk Mongyeong 323 CW14502 Chungnam Tehan151 CW15891 Gyeongbuk Mongyeong 324 CW15321 Chungnam Hongseong152 CW16058 Gyeongbuk Bonghuwa 325 CW14273 Chungbuk Guisan153 CW16650 Gyeongbuk Bonghuwa 326 CW15036 Chungbuk Danyang154 CW16963 Gyeongbuk Sangju 327 CW15318 Chungbuk Danyang155 CW16966 Gyeongbuk Sangju 328 CW16663 Chungbuk Buen156 CW15926 Gyeongbuk Sangju 329 CW14237 Chungbuk Buen157 CW16959 Gyeongbuk Sangju 330 CW16666 Chungbuk Yangdong158 CW16969 Gyeongbuk Sangju 331 CW17029 Chungbuk Okcheon159 CW16004 Gyeongbuk Seongju 332 CW17210 Chungbuk Okcheon160 CW15893 Gyeongbuk Seongju 333 CW17211 Chungbuk Okcheon161 CW16257 Gyeongbuk Seongju 334 CW17212 Chungbuk Okcheon162 CW15907 Gyeongbuk Seongju 335 CW14220 Chungbuk Okcheon163 CW15903 Gyeongbuk Seongju 336 CW16327 Chungbuk Omseong164 CW14645 Gyeongbuk Andong 337 CW16326 Chungbuk Omseong165 CW15291 Gyeongbuk Andong 338 CW15881 Chungbuk Omseong166 CW15303 Gyeongbuk Andong 339 CW14954 Chungbuk Omseong167 CW15295 Gyeongbuk Andong 340 CW15002 Chungbuk Jecheon168 CW15305 Gyeongbuk Andong 341 CW14286 Chungbuk Chungpyeong169 CW16598 Gyeongbuk Yangdeok 342 CW15879 Chungbuk Jincheon170 CW13295 Gyeongbuk Yangdeok 343 CW14293 Chungbuk Chungwon171 CW16605 Gyeongbuk YangYang 344 CW14967 Chungbuk Chungju172 CW16196 Gyeongbuk Yangju 345 CW16557 Chungbuk Chungju173 CW16926 Gyeongbuk Yangcheon 346 CW14989 Chungbuk Chungju

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using the external standard (i.e. based on calibration curves). The concentration of each of the six components (i.e. daidzin, glycitin, genistin, m-daidzin, m-glycitin, and m-genistin) was efficiently determined and the average values of triplicate HPLC analysis were recorded and used for all analysis.

Total soy-isoflavone content varied from 0.5 to 5.51 mg/g with an average of 2.78 mg/g. The most abundant soy-isoflavone in Korean wild soybeans was m-genistin (1.04 mg/g), whereas the least abundant soy-isoflavone was glycitin (0.0859 mg/g) (Figure 2).

Figure 3 shows the frequency distribution of total daidzin, genistein, and glycitin and total soy-isoflavone content for Korean wild soybeans. Frequency distributions of total glycitin, daidzin, and genistin departed from normal and showed positive skewness. However, total soy-isoflavone content was distributed normally. Soy-isoflavone content and composition in wild soybean from all nine Korean provinces was compared.

Statistically significant differences between Jeonnam and other provinces (i.e. Gyeongsang, Jeongbuk, Gyeongbuk, Chungnam, Gyeonggi, Gangwon, and Jeju) were found for total soy-isoflavone content. All Korean wild soybean accessions showed smaller differences regarding total soy-isoflavone contents when compared by provinces (Table 2).

To explore variations in soy-isoflavone contents in Korean wild soybeans, PCA was performed. The first two axes of the PCA explained 78.91% of the total variance. The PCA showed no distinct grouping in relation to soy-isoflavone content (Figure 4).

Among the glycosides, genistin was the most abundant soy-isoflavone with the average amount of 1.32 mg/g, followed by daidzin and glycitin having amounts of 0.86

and 0.279 mg/g, respectively. Among malonyl glycosides, maximum quantity was found for m-genistin (1.04 mg/g), followed by m-daidzin (0.712 mg/g) and m-glycitin (0.195 mg/g) (Table 3).

A general increasing trend was clearly visible when total soy-isoflavone content was laid out according to the accessions’ geographical locations using the longitude and latitude of collection sites. Soy-isoflavone content of accessions from the north part of the country was lower than that of accessions from southern parts and contents increased towards the southeast with minor variations (Figure 5). The total glycosides content was lower (20.4%) compared to malonyl glycosides (79.59%). Significant differences were observed in total daidzin, glycitin, and genistin contents among all Korean wild soybeans. Furthermore, the highest content of total soy-isoflavone component was found in genistin (1.393 mg/g), followed by daidzin (0.939 mg/g) and glycitin (0.250 mg/g). The highest total soy-isoflavone content was in an accession CW10110 (collected from Jeju, an island on the southeastern side of Korea) and the lowest content was in CW16009 (collected from Kangwon) (Table 2).

4. DiscussionSoy-isoflavones have assumed an important status in food and nutraceuticals because of their health benefits in prevention of certain types of cancers, cardiovascular diseases, and osteoporosis (Tepavcevic et al., 2010). Apart from their health benefits, they play an important role in various biological processes in plants, such as induction of nodulation during symbiosis, antimicrobial phytoalexins, allochemicals, and insect antifeedants, as well as stimulation of fungal spore germination in fungus (Ndakidemi and Dakora, 2003). The contents and compositions of soy-

0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 32.5 35.0 37.5 40.0 42.5 min

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Figure 2. HPLC chromatograms of six isoflavones in Korean wild soybean: (A) standard chromatogram of crude isoflavone, (B) Korean wild soybean seeds (CW16963); 1, daidzin; 2, glycitin; 3, genistin; 4, malonyl-daidzin; 5, malonyl-glycitin; 6, malonyl-genistin.

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isoflavone vary in different Glycine species and depend upon many factors, such as genotype, variety, location, year, and origin. To accelerate breeding programs for improved soy-isoflavone contents, it is necessary to identify accessions with high isoflavone contents, QTLs, and/or genes responsible for soy-isoflavone content. To determine the profile of wild soybeans from the Korean peninsula grown with the same environmental conditions, year, and location, the present study is a legitimate strategy. Exclusion of environmental effects can aid in making a rational decision in soybean breeding for higher and lower isoflavone content. In this study, the soy-isoflavone

content of wild soybean accession seeds collected from a wide range of habitats in the South Korean territory was determined.

Soy-isoflavone profiles of 346 Korean wild soybeans were compared and all six types of soy-isoflavones were detected. Korean soybeans have been previously reported to have a higher soy-isoflavone content (Lee et al., 2003, 2010; Kim et al., 2012). Minimum and maximum total soy-isoflavone content in Korean wild soybean accession seeds in our study was respectively 0.50 and 5.51 mg/g, which is quite close to previous studies on small-sized Korean soybeans (i.e. 0.81 to 4.78 mg/g; Kim et al., 2012). Korean soybeans have been shown to have a wider distribution of soy-isoflavone content (1.88 to 9.49 mg/g; Lee et al., 2003). It is beneficial to identify accessions of wild soybean with a wider distribution of soy-isoflavones based on explored genotypes. The range of soy-isoflavone content observed in this study suggested a combined effect of multiple alleles/QTLs (Pei et al., 2018).

This study showed that the maximum soy-isoflavone content in wild soybean seeds was collected from Jeju. The other soybean accessions from Korea exhibited mean soy-isoflavone content that varied nonsignificantly among the provinces, except for accessions from Jeonnam Province, which differed significantly from those of other provinces. However, once the provincial grouping was removed, differences were observed in total soy-isoflavone content (0.50 and 5.51 mg/g) in all Korean wild soybean accession seeds and plotted the contents according to longitude and latitude.

It is interesting to note that a trend in total soy-isoflavone content from the north to the southeastern

Table 2. Isoflavone content of 346 wild soybeans from 9 provinces of Korea.

Province nIsoflavone content (mg/g of soybean seed)

Elevation* Daidzin Glycitin Genistin M-daidzin M-glycitin M-genistin Total

Jeju 98 24 0.150 ± 0.08 0.071 ± 0.04 0.272 ± 0.11 0.789 ± 0.30 0.179 ± 0.06 1.121 ± 0.35 2.582 ± 0.77ab

Jeonnam 192 61 0.162 ± 0.07 0.082 ± 0.04 0.340 ± 0.13 0.836 ± 0.28 0.209 ± 0.09 1.277 ± 0.37 2.906 ± 0.85a

Gyeongnam 472 55 0.179 ± 0.08 0.095 ± 0.06 0.302 ± 0.11 0.702 ± 0.22 0.187 ± 0.07 1.090 ± 0.30 2.555 ± 0.70ab

Jeonbuk 449 29 0.145 ± 0.06 0.081 ± 0.03 0.301 ± 0.10 0.747 ± 0.24 0.201 ± 0.06 1.097 ± 0.32 2.571 ± 0.67ab

Gyeongbuk 187 66 0.156 ± 0.07 0.092 ± 0.04 0.264 ± 0.12 0.700 ± 0.21 0.200 ± 0.07 1.012 ± 0.38 2.426 ± 0.69b

Chungbuk 220 22 0.148 ± 0.08 0.081 ± 0.04 0.262 ± 0.10 0.682 ± 0.28 0.191 ± 0.07 1.025 ± 0.29 2.388 ± 0.74b

Chungnam 108 16 0.143 ± 0.08 0.091 ± 0.04 0.204 ± 0.10 0.649 ± 0.30 0.197 ± 0.06 0.902 ± 0.35 2.185 ± 0.78b

Gyeonggi 44 14 0.130 ± 0.07 0.098 ± 0.05 0.229 ± 0.10 0.664 ± 0.30 0.199 ± 0.07 0.962 ± 0.34 2.282 ± 0.79b

Gangwon 383 59 0.140 ± 0.08 0.084 ± 0.03 0.194 ± 0.10 0.640 ± 0.28 0.194 ± 0.06 0.883 ± 0.34 2.135 ± 0.79b

Total Average 346 0.150 ± 0.07 0.086 ± 0.04 0.263 ± 0.11 0.712 ± 0.11 0.195 ± 0.07 1.041 ± 0.34 2.448 ± 0.75

*Source: https://elevationmap.net/. Different superscript letters indicate significant differences among cultivars (P < 0.05).

0

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daidzinglycitin

genistin

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Figure 3. Frequency distribution of glycitin, daidzin, genistin and total isoflavone content in Korean wild soybeans.

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side of the country was observed. We assumed that this effect might be caused by temperature variations in natural habitats. As we move from the north to southeastern side of the country, slight differences in temperature occur, the northern habitats being colder than the southeastern side of the country. Previously, it was suggested that temperature and location are the factors that can influence soy-isoflavone content in soybeans (Tsukamoto et al., 1995). A similar trend can be visualized in previous results where accessions from Iksan (southeastern side of the country) had higher total soy-isoflavone contents as

compared to those located near northern areas (Lee et al., 2010). This new finding can contribute greatly in selection of accessions with higher soy-isoflavone content and the results can possibly be generalized when selecting wild soybean accessions based on geographic distribution. In addition, this trend in Korean wild soybeans has not been observed or reported in any previous studies (Lee et al., 2003, 2010; Kim et al., 2012).

The observed soy-isoflavone content variation in Korean wild soybeans (0.5–6.50 mg/g) grown in the same year and location and under the same environmental

Figure 4. Variance of Korean wild soybean accessions in isoflavone content explained by PCA: 1, Jeju; 2, Jeonnam; 3, Gyeongnam; 4, Jeonbuk; 5, Gyeongbuk; 6, Chungbuk; 7, Cheongnam; 8, Gyeonggi; 9, Gangwon.

Table 3. Content of total daidzin, total glycitin, and total genistein of 346 Korean wild soybeans from 9 provinces of Korea.

ProvinceIsoflavone content (mg/g of soybean seed)

Total daidzin Total glycitin Total genistin

Jeju 0.939 ± 0.01ab 0.250 ± 0.009d 1.393 ± 0.008ab

Jeonnam 0.998 ± 0.009a 0.291 ± 0.010b 1.617 ± 0.008a

Gyeongnam 0.881 ± 0.009abc 0.281 ± 0.010bc 1.393 ± 0.008ab

Jeonbuk 0.892 ± 0.008abc 0.282 ± 0.008c 1.398 ± 0.008ab

Gyeongbuk 0.857 ± 0.009abc 0.292 ± 0.009b 1.277 ± 0.009bc

Chungbuk 0.920 ± 0.01ab 0.280 ± 0.009c 1.329 ± 0.008bc

Chungnam 0.706 ± 0.01c 0.259 ± 0.009d 1.229 ± 0.009bc

Gyeonggi 0.793 ± 0.01bc 0.297 ± 0.009a 1.191 ± 0.009bc

Gangwon 0.780 ± 0.01bc 0.278 ± 0.008c 1.077 ± 0.010c

Average 0.8628 ± 0.01 0.279 ± 0.009 1.323 ± 0.090

Different superscript letters indicate significant differences among cultivars (P < 0.05).

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conditions showed much wider distribution than those reported for cultivated soybeans [G. max (L.) Merr.], American soybeans [1.18–4.22 mg/g (n = 30)] (Xu and Chang, 2008), Brazilian soybeans [0.57 to 1.88 mg/g (n = 13)] (Genovese et al., 2005), and Indian soybeans [0.76 mg/g (n = 4)] (Devi et al., 2008), excluding Korean soybeans [1.88-9.49 mg/g (n = 15)] (Lee et al., 2003).

In terms of soy-isoflavone composition and type, the maximum and minimum amounts were measured for m-genistin (2.31 mg/g) and glycitin (0.0859 mg/g). This observation was similar to a previous report on Korean black soybeans (Lee et al., 2010). Regarding total daidzin, glycitin, and genistin content, Korean wild soybeans had a similar trend as that previously observed in Korean black soybeans. In this study, genistin was the most abundant soy-isoflavone, followed by daidzin and glycitin; however,

in previous reports a similar trend was observed (Lee et al., 2010). In contrast, different trends have also been reported (Kim et al., 2012).

The accessions identified with higher and lower contents in our studies will be used to validate their phenotypes under different environmental conditions. Similar strategies were adopted by the Ministry of Agriculture, Forestry, and Fisheries for the development of a high soy-isoflavone content variety, Fukuibuki, in 2004 (Shimada et al., 2004). Furthermore, the information on genetic variations between high and low soy-isoflavone content accessions will increase knowledge regarding good combinations of alleles of the soy-isoflavone biosynthetic pathway affecting the seed soy-isoflavone content in future.

Absence of m-glycitin in an accession (CW12125) might have developed because of environmental effects

Figure 5. Geographical distribution of isoflavone content in Korean wild soybeans.

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or by natural mutation. This accession will be valuable in soybean breeding. Environment can affect the expression of soy-isoflavone content; however, the complete absence of m-genistin from this accession cannot be explained at this step and further studies are required. The origin of the accessions had an effect on soy-isoflavone contents, especially when comparing the total soy-isoflavone contents in Korea.

In conclusion, analysis of 346 Korean wild soybean seeds showed that among the Korean provinces no significant differences were present when mean values of total soy-isoflavone contents and their components were compared, except for Jeonnam Province. Korean wild soybeans followed an increasing trend in terms of total

soy-isoflavone content from the north to the southeast. The information generated in this report will greatly aid soybean breeding stratagems aimed at improving soy-isoflavone concentration in soybean seeds. The accessions with the highest and lowest contents and the accession missing m-glycitin might be used to identify unsolved genetics of soy-isoflavones in soybeans.

AcknowledgmentThis research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF), funded by the Ministry of Education (NRF-2015R1D1A1A09060925).

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