Profiling seed soluble sugar compositions in 1164 Chinese soybean accessions from major growing ecoregions

Jie Qi,Shengrui Zhng,Muhmm Azm,Abulwhb S.Shibu,Ahme M.Abelghny,Yue Feng,Yunyun Hui,Huoyi Feng,Yitin Liu,Ciyou M,Berhne S.Gebregzibher,Suprio Ghosh,Jing Li,Deyue Yu,Bin Li,Lijun Qiu,Junming Sun,*

a The National Engineering Research Center of Crop Molecular Breeding,Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

b The National Key Facility for Crop Gene Resources and Genetic Improvement/Key Laboratory of Germplasm and Biotechnology(MARA),Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

c MARA Key Laboratory of Soybean Biology(Beijing),Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China

d National Key Laboratory of Crop Genetics and Germplasm Enhancement,National Center for Soybean Improvement,Jiangsu Collaborative Innovation Center for Modern Crop Production,Nanjing Agricultural University,Nanjing 210095,Jiangsu,China

e Department of Agronomy,Bayero University,Kano 700001,Nigeria

f Crop Science Department,Faculty of Agriculture,Damanhour University,Damanhour 22516,Egypt

Keywords:Soluble sugars Soybean[Glycine max(L.)Merrill]UPLC-RID Natural variation Geographical distribution

ABSTRACT Soluble sugar is a key quality trait of soybean seeds.We developed rapid and economic extraction and quantification methods for seed soluble sugars using an ultra-high performance liquid chromatography system with a refractive index detector.We evaluated the soluble sugar compositions of 1164 soybean accessions collected from diverse ecoregions and grown in multiple locations and years.Total soluble sugar(TSS)content was influenced by accession type,year of cultivation,and ecoregion.The mean contents of fructose,glucose,sucrose,raffinose,stachyose and TSS were 3.31,5.21,55.60,6.60,35.47,and 106.19 mg g-1,respectively.The highest mean TSS content(108.71 mg g-1)was observed in accessions from Northern Region of China.Cultivars contained higher contents of sucrose,raffinose,and TSS,whereas landraces had a higher content of stachyose.Fourteen accessions with mean TSS contents＞130 mg g-1 were identified as elite soybean resources.TSS was correlated with sucrose,raffinose,stachyose,protein,oil and total tocopherol.The main soluble sugar components were correlated with latitude and longitude,indicating that the geographical origin of the accessions affected their seed soluble sugar compositions.The developed methods and elite identified accessions can be used in the food and feed industry and in soybean breeding programs aimed at improving soybean seed nutrition.

1.Introduction

Soybean[Glycine max(L.)Merrill]is a sourc e of protein,oil,carbohydrates,vitamins,inorganic salts,and micronutrients for humans and animals[1].Soybean seed carbohydrates are categorized into two forms:structural and non-structural.The latter is referred to as soluble sugar[2].The most abundant soluble sugar components in soybean seeds are sucrose and stachyose,followed by raffinose,glucose,and fructose[3].Sucrose,fructose,and glucose enhance the overall taste and sweetness of soy foods(soymilk,natto,tofu,etc.),whereas stachyose and raffinose are indigestible and cause flatulence and diarrhea in humans and monogastric animals.However,stachyose and raffinose act as Bifidus factors that improve intestinal flora and greatly reduce the risk of gastrointestinal infections and cardiovascular disease[4-7].

Soluble sugar contents are quantitative traits affected by genetic and environmental factors[8].Total soluble sugar(TSS)has been reported to be positively associated with altitude,diurnal temperature range,and photoperiod,but negatively associated with cumulative temperature and mean daily temperature[8],whereas raffinose and stachyose accumulations are influenced by genetic factors[5,9].To date,research on soluble sugars has used only a few accessions grown in limited locations and not representing the full genetic diversity of soluble sugars.Largescale evaluation of soybean germplasm across multiple years and locations would better reveal the genetic diversity of soluble sugar content of soybean seeds.However,because currently available soluble sugar quantification methods are laborious and timeconsuming,quantifying soluble sugars remains a challenge in large-scale samples.The present study aimed to i)develop an accurate,efficient,and economical method for extracting and quantifying soluble sugars,ii)characterize variation in soluble sugars in Chinese soybean germplasm,iii)investigate the correlations among soluble sugar components and other seed nutritional characteristics,and iv)visualize the geographical distribution of soluble sugars in China.

2.Materials and methods

2.1.Plant materials and field experiments

A set of 1164 Chinese soybean accessions was used,including 923 landraces and 241 cultivars selected from a core collection of 2794 accessions developed from 23,587 accessions conserved in the National Crop Gene Bank of China.The selected accessions accounted for 41.7% of the genetic diversity of the entire core collection[10].These accessions were collected from China’s three major soybean ecoregions:242 from the Northern Region(NR),432 from the Huang Huai Hai Valley Region(HR),and 490 from the Southern Region(SR),with the aim of capturing as much phenotypic and geographic diversity as possible.Detailed information on the accessions used in this study is presented in Table S1.All accessions were planted at Changping,Beijing(40°13′N,116°12′E)and Sanya,Hainan(18°24′N,109°5′E)in 2017 and 2018.The experiment was laid out in a randomized incomplete block design,with the two planting locations as replications.All details of field experiments have been previously reported[11-14].At each location,separate randomization was performed,and seeds of each accession were sown in a single-row plot 3 m in length with 0.5 m spacing between rows and 0.1 m between plants within rows.After emergence,plants were thinned to leave only uniform healthy plants.During land preparation,organic fertilizer was applied at the rate of 15 t ha-1,providing 30 kg N ha-1,60 kg P ha-1,and 50 kg K ha-1.Weeds were controlled by a postemergence application of 2.55 L ha-1of acetochlor,as well as hand weeding during the growing season.All accessions were harvested manually at maturity in all locations.After harvesting,all the seeds of each accession were pooled and about 200 seeds were randomly selected from each accession for determination of seed quality components.

2.2.Standard solutions and calibration curves

Fructose,glucose,sucrose,melibiose,raffinose,and stachyose standards obtained from Shanghai Yuanye Bio-Technology Co.,Ltd.(Shanghai,China)were used to prepare standard solutions at various concentrations(0.1,0.3,0.5,0.8,1.0,2.0,4.0,and 8.0 mg mL-1)using 50% acetonitrile(Thermo Fisher Scientific,Pittsburgh,PA,USA).To prepare working standard solutions,500μL of each concentration of the standard solutions was diluted with 200μL of acetonitrile.This procedure was performed to guarantee that the working standard solutions were treated in the same manner as the soybean samples,reducing test error and matrix effects.Calibration curves were measured by injecting the working standard solutions in triplicate.

2.3.Soluble sugar extraction and determination

Soybean seeds were ground into a fine powder using an electric grinder(Retsch ZM100,Φ=1.0 mm,Haan,Germany).For the extraction of soybean soluble sugar,three parameters were optimized.First,the concentration of acetonitrile was optimized to identify the best concentration for soybean soluble sugars.The evaluated concentrations of acetonitrile were 0,50%,60%,65%,70%,75%,and 80%(v/v).Second,the incubation time during shaking was evaluated(1,2,4,8,and 12 h),and third,ultrasonic extraction was investigated at four time periods(10,30,60,and 120 min).The initial extraction procedure was devised as follows:100 mg of soybean powder was mixed with 1 mL of 50% acetonitrile and shaken at 200 r min-1for 8 h at room temperature,followed by centrifugation at 12,000 r min-1for 10 min at 20 °C.Five hundred microliters of the supernatant were mixed with 200μL of acetonitrile in a fresh 1.5-mL centrifuge tube and left at room temperature to precipitate remaining proteins.The supernatant was centrifuged at 12,000 r min-1for 10 min at 20 °C,and then filtered using a syringe filter(0.22μm),and collected in a 2-mL bottle.

Soluble sugars were quantified by ultra-high-performance liquid chromatography with a refractive index detector(UPLC-RID)(H-Class,Waters,Milford,MA,USA).Separation of six soluble sugars was performed on the Acquity BEH amide column(2.1×50 mm,1.7μm,Waters)and the elution buffer contained acetonitrile with 0.2%(v/v)triethylamine.To produce the optimal chromatographic conditions,several column temperatures(30,50,and 85 °C),acetonitrile concentrations(60%,70%,75% and 80%)and flow rates(0.2,0.3 and 0.4 mL min-1)were evaluated.The injection volume was 1.0μL.

2.4.Method validation parameters

The method was validated based on selectivity,linearity,precision,and accuracy.Selectivity was determined when samples were spiked with standards and the area of the corresponding analytes increased proportionally.The linearity of the method was evaluated by calculating the linear regression coefficient of the calibration curve for each component.Recovery was used to assess the method’s accuracy.For recovery experiments,samples were spiked with soluble sugar standards at three(low,medium,and high)concentrations(n=3).Recovery was computed as followed:

Injection precision was determined by injecting the standard solution of each soluble sugar component three times.Relative standard deviations(RSD)of retention time and peak area were used to assess the repeatability and reproducibility of the method.Limits of detection(LOD)and limits of quantification(LOQ)of the chromatographic conditions were determined based on the response value and slope of each regression equation at signalto-noise ratios(S/N)of 3 and 10,respectively.

2.5.Determination of other seed quality traits

The protein and oil contents were measured by Fourier transform near-infrared spectroscopy(MPA,Bruker Fourier,Rheinstetten,Germany).Fatty acid contents were determined using gas chromatography(GC-2010,Shimadzu Inc.,Kyoto,Japan).Isoflavone and tocopherol contents were quantified using an Agilent 1260 HPLC system(Agilent,Santa Clara,CA,USA).The detailed procedures used for oil,protein,fatty acids,isoflavone,and tocopherol determination and their data collection have been previously reported[1,11,12,13].

2.6.Statistical analysis

Analysis of variance(ANOVA)for seed soluble sugar composition was fitted with PROC GLM in SAS 9.2(SAS Institute,Cary,NC,USA).Three separate ANOVAs were computed to estimate the effects of accession,ecoregions of origin,and accession type on soluble sugar contents.Accession,ecoregion of origin,and accession type were treated as fixed effects in each ANOVA,whereas location was nested in year and treated as random.Pearson’s correlation coefficient(r)and boxplots were calculated using R statistical software version 3.6.3(R Foundation for Statistical Computing,Vienna,Austria).Geographical distribution maps of seed soluble sugars were drawn with ArcGIS Pro 2.7(Esri,Redlands,CA,USA).

3.Results

3.1.Development of rapid extraction and detection methods for soluble sugars

The optimum extraction conditions chosen employed 50% acetonitrile as the solvent,followed by 8 h of shaking at room temperature(Tables S2,S3).All other parameters remained unchanged as described in Section 2.3.Soluble sugars were quantified in 11 min at a flow rate of 0.4 mL min-1by UPLC-RID.The column and flow cell temperatures were held at 85 °C and 40 °C,respectively.The mobile phase consisted of 80% acetonitrile with 0.2% trimethylamine(Figs.S1,S2).

The extraction and detection methods were validated for linearity(R2greater than 0.999),LOD(0.030-0.231 mg mL-1),LOQ(0.0 97-0.763 mg mL-1),recovery(84.16%-103.53%)and precision(RSD＜2.40%)(Table 1).

3.2.Natural variation of soluble sugar contents in soybean accessions

Among the individual soluble sugars,sucrose takes the most part of TSS(55%),followed by stachyose(33%).The other three soluble sugars(fructose,glucose,raffinose)take less parts of TSS(＜10%)(Table S4).Both individual and total soluble sugar contents varied significantly(Table S5)and exhibited bell-shaped distributions(Fig.S3).Fructose showed the highest coefficient of variance(CV),while raffinose shows the lowest one,suggesting a high degree of variation for fructose but low degree variation for raffinose in 1164 Chinese soybean accessions(Table S4).

3.3.Effects of accession type and ecoregion on soybean seed soluble sugar content

Differences(P＜0.05)were observed among years,accession types,and ecoregions,as well as a significant effect for the year×ecoregion interaction for soluble sugar components other than fructose and glucose(Table S5).Between accession types,higher levels of sucrose(60.57 mg g-1),raffinose(6.88 mg g-1),and TSS(111.30 mg g-1)were observed in cultivars,whereas landraces contained higher contents of stachyose(35.77 mg g-1)(Fig.1A-F).Among the three major ecoregions of China,the highest levels of fructose(3.49 mg g-1),glucose(5.46 mg g-1),sucrose(57.89 mg g-1),raffinose(6.93 mg g-1),and TSS(108.71 mg g-1)were observed in the NR accessions,whereas the highest levels of stachyose(36.49 mg g-1)were observed in the SR accessions(Fig.1G-L).

3.4.Correlations between soluble sugars and other seed quality traits

Pearson’s correlations among soybean seed nutritional quality characteristics are shown in Fig.S4.TSS content was correlated with sucrose(r=0.90***),raffinose(r=0.55***),and stachyose(r=0.31***).For individual soluble sugars,fructose and glucose were positively correlated(r=0.86***),whereas negative correlations were observed between sucrose,raffinose,and stachyose.However,raffinose was positively correlated with stachyose(r=0.36***)and sucrose(r=0.36***).

For other seed nutritional quality characteristics,TSS was correlated with protein(r=-0.34***),oil(r=0.30***),stearic acid(r=0.25***),glycitin(r=-0.27***),malonylglycitin(r=-0.25***),and total tocopherol(r=0.25***).Glucose and fructose showed positive correlations with daidzein,genistein,andα-tocopherol.

3.5.Geographic variation of soybean seed soluble sugar compositions

Mean individual and total soluble sugar contents across locations and years were correlated with geographical factors:the latitude,longitude,and altitude of their corresponding regions of origin(Table S6).Individual and total soluble sugars except for raffinose and stachyose showed positive correlations with latitude and longitude.

The geographical distribution maps of sucrose and TSS contents clearly showed that NR accessions were richer in these components than those from SR(Fig.2A,C).We identified hotspot points in the NR(Nen River and Songhua River basin)with higher levels of sucrose and TSS contents(Fig.2A,C).We also identified hotspot points in the SR(Yangtze River and Lancang River basins)with high levels of sucrose and TSS contents(Fig.2A,C).

In contrast,stachyose content showed high negative correlations with latitude(P＜0.001),longitude(P＜0.001)and altitude(P＜0.01)(Table S6).Stachyose content in the SR accessions was higher than that in the NR(Fig.2B).Low-altitude and-latitude areas such as Sichuan Basin,Leizhou Peninsula,and Hainan Island were identified as hotspots,in which the accessions accumulated higher levels of stachyose(Fig.2B).

Table 1Assessment of analytical method in terms of linear equation and interval,limits of detection and quantification,coefficient of determination,and intra-day,inter-day and recovery reproducibility.

Fig.1.Variation in individual and total soluble sugar contents in soybean landraces and cultivars(A-F),and three ecoregions of origin(G-L).NR,the Northern Region of China;HR,the Huang Huai Hai Valley Region of China;SR,the Southern Region of China.Different lowercase letters(a,b,and c)indicated differences at P＜0.05.

3.6.Identification of elite soybean accessions

We identified 14 elite soybean accessions(10 from SR and 4 from HR)with mean TSS content of more than 130.00 mg g-1(Table S7),including 11 landraces and 3 cultivars.Similarly,we identified 13 accessions(10 from SR and 3 from HR)with mean sucrose content of more than 80.00 mg g-1(Table S8),including 11 landraces and 2 cultivars.We identified 12 landraces with mean stachyose concentrations of more than 45.00 mg g-1,originating in SR(Table S9).

Fig.2.Geographical distribution of individual and total soluble sugar contents in soybean seeds mapped by the accession’s region of origin.(A)Sucrose;(B)stachyose;(C)total soluble sugar.The three ecoregions(NR,Northern region;HR,Huang Huai Hai valley region;and SR,Southern region)are indicated with red lines.(The maps were downloaded at the website http://bzdt.ch.mnr.gov.cn,and the map content approval number is GS(2019)1671.)

4.Discussion

We developed and optimized extraction and quantification methods for the determination of fructose,glucose,sucrose,melibiose,raffinose,and stachyose in soybean seeds.In this study,proteins were precipitated by direct addition of acetonitrile without further enrichment or purification processes,making the extraction method simple and efficient(Tables S2 and S3).Various extraction methods have been developed for soybean soluble sugars[4,15,16].However,these methods are relatively complex and time-consuming.They also have limitations,such as enriching and purifying soluble sugars using C18 cartridges to remove proteins,which reduce their efficiency and repeatability.Also,heating at 50 °C or 60 °C during extraction to induce protein denaturation can coat soluble sugars and reduce extraction efficiency.

In this study,soluble sugars were quantified in 11 min at a flow rate of 0.4 mL min-1by UPLC-RID.Methods for quantitating soluble sugars in fruits by high-performance liquid chromatography with evaporative light scattering detection(HPLC-ELSD)and UPLC-ELSD have been published[17,18].However,their retention times and flow rates are 10 to 15 times longer and 2.5 times higher than those of our approach.Our extraction and detection methods are simple,rapid,and economical in comparison with those of previous studies.

Individual and total soluble sugar contents showed wider ranges in the 1164 Chines soybean accession than that reported in previous studies[8,19].This wide range of diversity shows the value of large-scale germplasm screening.In previous studies[11,12,14],landraces contained higher isoflavone,linoleic acid,linolenic acid,and folate contents than cultivars.Consistently,in this study,landraces also contained higher concentrations of stachyose than cultivars(Fig.1E),a finding that may be attributed to soybean seed environmental adaptation[1,20]and that also shows that soybean breeding activities may have led to the modification of seed quality traits.Stachyose and isoflavone function in plant adaptation to various stresses[21].Thus,higher stachyose and isoflavone contents could enable landraces to withstand harsh environmental conditions and improve their adaptation.

The differences in individual and total soluble sugar concentrations across the three ecoregions might be attributed to genetic and environmental factors.Lower temperature favors soluble sugar accumulation,according to Song et al.[8].However,their experiments were conducted in the original ecoregions of their accessions,whereas we examined accessions both within and outside their original ecoregions.The accessions from NR contained the highest sucrose and TSS contents among the three ecoregions even when grown outside of their original ecoregions.This founding would allow soybean breeders to broaden the range of germplasm utilization.

The correlation analysis among individual and total soluble sugars revealed the highest correlation between TSS and sucrose,followed by glucose and fructose.TSS was also positively correlated with raffinose and stachyose.These results showed that sucrose,raffinose,and stachyose influenced TSS accumulation,confirming earlier findings[1,22].Sucrose content showed a significant and positive correlation with raffinose,that may be because sucrose could be catalyzed by raffinose synthase to directly produce raffinose in soybean[23].

We investigated the interrelationships between soluble sugars and other seed nutritional characteristics with the aim of improving soybean breeding targeted at multiple quality traits.TSS was significantly and positively associated with oil,stearic acid,and total tocopherol contents.Stearic acid is important for oil stability,and tocopherols prevent heart diseases and cancer and help in strengthening the immune system[24].Glucose and fructose had positive associations with daidzein,genistein,andα-tocopherol.Daidzein and genistein are the aglycone forms of isoflavone that help prevent hormone-related cancers[25].Among the tocopherols,α-tocopherol has the highest vitamin E activity for human[24].The significant correlations between soluble sugars with other quality traits may make it possible to breed soybean cultivars with both higher soluble sugar contents and other favorable quality traits.

Geographical distribution maps of individual and total soluble sugar contents showed that accessions from higher latitudes were adapted to low temperature,which facilitated oil,sucrose,and TSS accumulation,whereas accessions from lower latitudes were adapted to high temperature,facilitating protein and stachyose accumulation,in agreement with previous studies(Figs.2,S5)[5,8,9,11].The hotspots in the SR(Yangtze River and Lancang River basins)with higher levels of sucrose and TSS(Fig.2A,C)might be attributed to vegetable-type soybean consumption habits and climatic factors in this region[20].In the Yangtze River basin,there are many vegetable-type soybean accessions called Maodou,which are harvested at the R6-R7 stage[26].Long-term selection of vegetable-type soybean varieties grown in these areas has resulted in a subtle preference for fresh taste quality.In the Lancang River basin,there are numerous mountains,with the daily cumulative temperature generally low throughout the year,favoring sucrose and TSS accumulation.

Soybean germplasm resources cultivated in China are abundant and diverse and have been incorporated into a complex planting system.It is of great importance to evaluate the seed quality traits of soybean accessions in diverse ecological regions to select elite accessions for soybean breeding and seed quality improvement.The variations found in the multiple ecological regions make it possible to select highly adapted germplasm with higher soluble sugar contents in different locations.The availability of soybean accessions with higher sucrose or stachyose contents will not only assist breeders in selecting elite parents for breeding but also provide new opportunities for food industries to manufacture soybean products based on consumer demand.

CRediT authorship contribution statement

Jie Qi:Investigation,Data curation,Visualization,Writingoriginal draft.Shengrui Zhang:Supervision,Conceptualization,Visualization,Writing-review & editing.Muhammad Azam:Resources,Writing-review & editing.Abdulwahab S.Shaibu:Resources,Writing-review & editing.Ahmed M.Abdelghany:Resources.Yue Feng:Resources.Yuanyuan Huai:Resources.

Huoyi Feng:Resources.Yitian Liu:Resources.Caiyou Ma:Resources.Berhane S.Gebregziabher:Resources.Suprio Ghosh:Resources.Jing Li:Resources.Deyue Yu:Supervision,Conceptualization.Bin Li:Supervision,Conceptualization,Visualization,Writing-review&editing.Lijuan Qiu:Supervision,Conceptualization,Visualization,Writing-review & editing.Junming Sun:Funding acquisition,Supervision,Conceptualization,Visualization,Writing-review & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by the National Key Research and Development Program of China(2021YFD1201605),the National Natural Science Foundation of China(3216114303 and 32001574),and Agricultural Science and Technology Innovation Program of Chinese Academy of Agricultural Sciences.

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.04.015.