Association of seed and agronomic traits with isoflavone levels in ...

Association of seed and agronomic traits with isoflavone levels in soybean Sheila E. Murphy1, Elizabeth A. Lee1, Lorna Woodrow2, Philippe Seguin3, Jagdish Kumar4, Istvan Rajcan1, and Gary R. Ablett5, 6 1

Dept. of Plant Agriculture, Univ. of Guelph, Guelph, Ontario, Canada N1G 2W1; 2Agriculture and Agri-Food Canada, 2585 Highway 20 East, Harrow, Ontario, Canada N0R 1G0; 3Department of Plant Science, McGill University, Ste. Anne de Bellevue, Quebec, Canada H9X 3V9; 4Hendrick Seeds, 11791 Sandy Row, Inkerman, Ontario K0E 1J0; and 5Ridgetown Campus, Univ. of Guelph, Ridgetown, Ontario, Canada N0P 2C0. Received 5 August 2008, accepted 9 February 2009. Murphy, S. E., Lee, E. A., Woodrow, L., Seguin, P., Kumar, J., Rajcan, I. and Ablett, G. R. 2009. Association of seed and agronomic traits with isoflavone levels in soybean. Can. J. Plant Sci. 89: 477
484. Soybean, Glycine max (L.) Merr., seeds contain isoflavones, compounds with potential human health benefits. This study investigated the association of seed and agronomic traits with isoflavone level in a genetically diverse group of soybean genotypes to provide more information for cultivar development. F4:7 lines derived from several crosses were grown in four locations in 2005 and six locations in 2006 across Ontario and Quebec. Seed protein, oil and isoflavone contents were measured using near-infrared reflectance (NIR) on a plot basis. Seed yield was determined at 13% moisture and days to maturity (R8) were recorded. GGE genotype-bytrait biplots were generated to describe the relationships among all variables. Isoflavone content was not correlated with yield, indicating that potential exists for development of high or low isoflavone cultivars without sacrificing yield. Isoflavone content was negatively correlated with protein content, however high isoflavone lines were identified with moderate protein content. Isoflavone content was correlated with maturity suggesting that delayed planting and/or the use of later maturing varieties could be a successful strategy to increase isoflavone content. The results of this study support the potential for the development of either high or low isoflavone soybean cultivars with acceptable agronomic and seed quality traits. Key words: Soybean, isoflavone, protein, oil, yield, maturity Murphy, S. E., Lee, E. A., Woodrow, L., Seguin, P., Kumar, J., Rajcan, I. et Ablett, G. R. 2009. Relation des caracte`res grainiers et agronomiques avec la concentration d’isoflavones chez le soja. Can. J. Plant Sci. 89: 477
484. Les graines du soja, Glycine max (L.) Merr., contiennent des isoflavones, compose´ susceptible d’avoir des effets be´ne´fiques sur la sante´. L’e´tude examinait les liens qui existent entre la concentration d’isoflavones et les caracte`res grainiers et agronomiques dans un groupe de varie´te´s de soja ge´ne´tiquement varie´, dans le but de fournir plus d’informations en vue de cre´er de nouveaux cultivars. Les ligne´es F4:7 issues de plusieurs croisements ont e´te´ cultive´es a` quatre endroits en Ontario et au Que´bec, en 2005, et a` six, en 2006. La teneur en prote´ines, en huile et en isoflavones des graines a e´te´ e´tablie par re´flectance dans le proche infrarouge, dans les parcelles. Le rendement grainier a e´te´ mesure´ a` 13 % de teneur en eau et on a enregistre´ le nombre de jours jusqu’a` maturite´ (R8). L’analyse GGE par double projection du ge´notype et des caracte`res a permis de de´crire les liens entre les variables. La concentration d’isoflavones n’est pas corre´le´e au rendement, signe qu’on pourrait cre´er des cultivars a` faible ou a` forte concentration d’isoflavones sans que le rendement en souffre. La concentration d’isoflavones pre´sente une corre´lation ne´gative avec la teneur en prote´ines, mais les ligne´es produisant beaucoup d’isoflavones donnent une concentration moyenne de prote´ines. La concentration d’isoflavones est corre´le´e a` la maturite´, si bien que retarder la plantation ou recourir a` des varie´te´s plus tardives pourrait eˆtre une bonne strate´gie pour accroıˆ tre la teneur en isoflavones. Les re´sultats de cette e´tude confirment qu’on pourrait de´velopper des cultivars de soja a` forte ou a` faible teneur en isoflavones posse´dant des caracte`res agronomiques et grainiers acceptables. Mots cle´s: Soja, isoflavone, prote´ines, huile, rendement, maturite´

Soybean, Glycine max (L.) Merr., is the world’s most important oilseed crop. Research has shown that the consumption of soy-foods has a positive impact on human health. This is, in part, due to isoflavones, which have potential health benefits in the prevention of heart

6

To whom correspondence should be addressed (e-mail: [email protected]). This work was submitted by Sheila E. Murphy in partial fulfilment for the Master’s degree from the Department of Plant Agriculture, at the University of Guelph.

Abbreviations: NIR, near-infrared reflectance; RIL, recombinant inbred line 477

478 CANADIAN JOURNAL OF PLANT SCIENCE

disease, specific cancers, osteoporosis, and treatment of post-menopausal symptoms (Setchell 1998). Isoflavones are naturally occurring, biologically active compounds, which are synthesized as secondary plant metabolites through the phenylpropanoid pathway. Within the plant isoflavones encourage infection and nodulation by Bradyrhizobium (Kosslak et al. 1987) enabling the plant to fix atmospheric nitrogen. Isoflavones are also involved in disease resistance responses to pathogens, specifically Phytophthora sojae, the organism responsible for Phytophthora root rot (Rivera-Vargas et al. 1993). The development of cultivars with increased isoflavone content may be one strategy to help meet the demand generated by public interest in soy-foods with enhanced health benefits. Such cultivars could increase the use of soybeans in the functional food market and may offer the potential for premium-based contract production to soybean producers. Successful cultivar development involves understanding the interrelationships among crop traits, both agronomic and seed attributes. Positive associations (Primomo et al. 2005; Seguin et al. 2004; Wang et al. 2000; Yin and Vyn 2005), negative associations (AlTawaha and Seguin 2006) and no associations (Charron et al. 2005) have been reported between total isoflavone concentration and seed yield. Wang et al. (2000) observed differences in total isoflavone concentration among maturity groups; however, a significant correlation between total isoflavone content and days to maturity was not found. Primomo et al. (2005) found that recombinant inbred lines (RILs) classified as high and intermediate isoflavone types matured significantly later than those with lower levels of seed isoflavone. In one of three populations the RILs classified as high and intermediate had significantly lower protein content than the low phenotypic classes, suggesting a negative relationship between these traits (Primomo et al. 2005). Yin and Vyn (2005) found a strong positive relationship of total isoflavone with seed yield, while oil and protein concentrations remained unchanged as yield increased. Charron et al. (2005) reported a moderate, negative correlation between total seed isoflavone and oil, but no significant correlation between total seed isoflavone and protein. Several studies have shown a small to moderate negative association between isoflavone levels and seed protein (Chiari et al. 2004; Seguin et al. 2004; AlTawaha and Seguin 2006). Overall, existing evidence on the relationships between total seed isoflavone content and other traits is conflicting. Research that includes a wide range of genetic material grown across a range of environments may provide additional insight into the association between total isoflavone and other agronomic and seed traits in soybean. This study investigated the relationship between total seed isoflavone content and protein, oil, yield, and maturity, with information from diverse genetic material over 10 environments. Employing

GGE genotype-by-trait biplot analysis assisted in fully understanding the interrelationships among these traits. MATERIALS AND METHODS Plant Material Lines were developed for this study to provide a wide range of isoflavone levels across diverse genotypes. Crosses were made in 2000 in controlled environment growth rooms in the Crop Science Building at the University of Guelph Campus, Guelph, Ontario. The F1 generation was space-planted at Ridgetown Campus, University of Guelph, in the spring of 2000. The F2 and F3 generations were grown in Costa Rica during the fall of 2000 and winter of 2001 using modified single seed descent and with populations sizes around 500 plants per cross In the spring of 2001, roughly 800 F4 seeds per cross were planted at the Ridgetown Campus. Approximately 150 F4 plants per cross were harvested at random to be grown in F4:5 single plant progeny rows in 2002. Progeny rows were grown at Ridgetown in single rows 5 m in length with 43 cm row widths. Lines were selected on the basis of maturity and general agronomic characteristics for further evaluation. In 2003, single rep yield trials of the F4:6 lines were conducted at two locations in southwestern Ontario. Seventy-five lines were tested again in 2-rep F4:7 yield trials in 2004 at two locations in southwestern Ontario. All trials in 2003 and 2004 were seeded into five-row plots, 5 m in length with 43 cm row widths. These trials were harvested with standard plot combines with yield, maturity, height, lodging, oil and protein levels being measured. The F4:7 lines grown in 2004 were analyzed for total isoflavone content and classified as high, intermediate or low, based on their relative isoflavone content within each population. Two groups of genotypes were selected from these crosses and treated as separate experiments. The first group (Group A) included 20 F4:7 lines from 10 of the crosses made in 2000. One ‘‘high’’ isoflavone entry and one ‘‘low’’ isoflavone entry from each cross was included. The 11 parents, comprising seven commercial cultivars and four plant introductions, were included in the analysis for a total of 31 entries in Group A. The second set of genotypes (Group B) included 15 F4:7 lines from each of two crosses, RCAT Angora (Ablett and Tanner 1993), a high isoflavone cultivar, and CK-01, a commercial cultivar, and Heinong35, a Chinese plant introduction, and RCAT Angora. Five lines of each class (high, intermediate, low) from each cross were included in the trial. The parents and two commercial checks, OAC Kent and Crown, were included in the analysis for a total of 35 entries in Group B. Field Experiments In 2005, each genotype was planted at four locations in southern Ontario, Canada: Ridgetown, Exeter, Inwood and Woodslee. In 2006, the lines were planted in the same locations in southern Ontario with the addition of

MURPHY ET AL. * SOYBEAN ISOFLAVONE CONTENT

two eastern Canadian locations: Inkerman, Ontario, and Ste. Anne de Bellevue, Quebec. The locations were chosen to be unique in agronomic factors affecting growth, including soil type, management, and climate. Each location was, however, suitable for the maturity ratings of the selected plant material and in total cover a major soybean producing region of Canada. A randomized complete block design was used at all locations. Group A was replicated twice and Group B was replicated three times. All plots were cultivated using recommended agricultural practices. Plot size varied at locations based on the management practices in use, but in all cases bordered plots were used. Fertilizer was added at recommended rates where required. Conventional herbicide programs were used at each location and the plots were kept weed free throughout the growing season. Agronomic Data Collection Days to maturity (R8) was recorded according to Fehr et al. (1971). All locations were harvested with a selfpropelled plot combine. Seed moisture was determined and yields were adjusted to 13% moisture. A seed sample was taken from the harvested material of each plot for seed constituent analysis. Seed protein, oil and isoflavone contents were measured using near-infrared reflectance (NIR) on a FOSS NIRSystems near infrared spectrophotometer, Model 6500 (FOSS NIRSystems, Inc., Laurel, MD) at Agriculture and Agri-food Canada, Harrow, Ontario. Isoflavone Analysis Whole seed samples were stored at 108C until preparation of the ground material for analysis. A 30
35 g seed sample from each plot was ground to a fine powder in a water-cooled FOSS Knifetec 1095 sample mill equipped with a sharp blade (FOSS NIRSystems, Eden Prairie, MN). The ground samples were transferred to a resealable plastic bag. The grinder chamber and other grinder parts were thoroughly cleaned to prevent carryover between samples. Ground samples were tempered at instrument room temperature in the plastic sample bags for 24 h prior to scanning. After all the samples had been scanned and spectra obtained, the scan files were standardized and the results were predicted. Total isoflavone content was predicted using a model constructed at Agriculture and Agri-Food Canada, Harrow, ON, and used for analysis of commercial soybean samples and historical cultivars (Morrison et al. 2008). Briefly, the model was constructed using laboratory reference data obtained by reverse phase HPLC separation and electrochemical detection of the genistein, daidzein, glycitein and the respective glucoside conjugates following extraction and saponification following AOAC Official Method 2001.10 (AOAC International 2003). Sample replicate values exhibited standard deviations consistent with AOAC

479

specifications. Total isoflavone was obtained by summing the aglycone equivalent values of the six constituent isoflavone compounds. The total isoflavone value for each sample was used to construct the predictive model using soybean samples from multiple seasons and multiple locations across a wide range of isoflavone contents. The samples from the 2005 and 2006 seasons were predicted from the stored spectra using the same model equations. Equation statistics for the total isoflavone model used in this study were as follows: mean 2260 mg kg1; SD, 815; SEC, 240; RSQ, 0.9134; SECV, 304; 1-VR, 0.8608. Statistical Analysis A combined analysis of variance was computed separately for each of the groups, A and B, to estimate the main effects of the environments, genotypes, and their interactions on total isoflavone content, oil content, protein content, days to maturity and yield in soybean. Genotypes were considered fixed effects, and replications and environment were considered random effects. The analysis of variance, least square means, and standard errors for each trait were calculated using the PROC MIXED procedure of the SAS software (release 9.1.3) (SAS Institute, Inc. 2003). A test for outliers was conducting using Lund’s (Lund 1975) test of studentized residuals and the General Linear Model (GLM) procedure in SAS (release 9.1.3) (SAS Institute, Inc. 2003). Five outliers were identified in Group B and removed from the analyses. Pairwise comparisons of class means were made using Tukey’s multiple means comparison method with a Type I error of 0.01 used for all statistical comparisons. GGE genotype-by-trait biplots were generated for Group A and Group B using GGE biplot software (Yan 2001). RESULTS The analysis of variance indicated that for all traits in both experiments the effects of environment, genotype and the interaction of genotype by environment were significant. Generally, in this study, environments which were warmer and drier resulted in less seed isoflavone levels across all genotypes than those environments which were cooler and wetter, consistent with previous studies (Tsukamoto et al. 1995; Caldwell et al. 2005; Lozovaya et al. 2005). Mean isoflavone level ranged from 1600 to 3700 mg g1 across the 10 environments. The genotypes in this study covered a wide range for all traits. In Group A, total isoflavone content of the 31 entries ranged from 1729 to 3514 mg g1, maturity from 112 to 127 d, oil content from 18.8 to 21.7%, protein content from 40.0 to 44.8% and yield from 2900 to 4515 kg ha1 (Table 1). In Group B, total isoflavone content of the 35 genotypes ranged from 1576 to 3509 mg g1, maturity ranged from 113 to 130 d, oil content from 19.3 to 21.3%, protein content from 40.8 to 44.5% and yield from 3282 to 4458 kg ha1 (Table 2).

480 CANADIAN JOURNAL OF PLANT SCIENCE Table 1. Means and standard errors of isoflavone content, maturity, oil content, protein content and yield of 31 soybean (Glycine max L. Merr) genotypes belonging to Group A grown in 10 environments in 2005 and 2006 Entry number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31

Pedigree RCAT Angora  OAC Arthur RCAT Angora  OAC Arthur RCAT Angora  PRO 28-53 RCAT Angora  PRO 28-53 RCAT Angora  Ivory RCAT Angora  Ivory RCAT Angora  CK-01 RCAT Angora  CK-01 RCAT Angora  S20-F8 RCAT Angora  S20-F8 RCAT Angora  Tsurukogane RCAT Angora  Tsurukogane RCAT Angora  DongNong42 RCAT Angora  DongNong42 Heinong35  RCAT Angora Heinong35  RCAT Angora Heinong35  Ivory Heinong35  Ivory RCAT Appin  Suzumaru RCAT Appin  Suzumaru RCAT Angora OAC Arthur PRO 28-53 Ivory CK-01 S20-F8 Tsurkogane DongNong42 Heinong35 RCAT Appin Suzumaru

Isoflavone classificationz

Isoflavone (mg g1)

Maturity (days)

Oil (%)

Protein (%)

Yield (mt ha1)

L H L H L H L H L H L H L H L H L H L H P P P P P P P P P P P Mean SEM

2165 2956 2358 2987 2094 2538 2703 3497 2830 3514 2725 3269 2670 2972 2099 2432 1729 2139 1882 2127 3219 2670 2329 2061 2495 1941 2558 2196 1955 2293 2143 2501 203

117 121 123 125 124 126 122 126 126 125 124 119 127 118 121 113 118 118 124 113 126 112 114 120 122 119 124 118 115 122 120 121 4

20.9 21.0 21.2 21.7 20.7 20.9 20.6 20.5 21.1 21.2 20.3 21.4 20.4 20.4 20.9 20.5 19.8 19.6 20.3 20.2 21.2 20.3 21.6 20.7 20.4 21.3 18.8 19.3 19.5 20.7 20.0 20.6 0.3

42.2 40.4 41.6 40.0 42.9 43.0 42.2 42.3 41.4 40.4 41.8 40.3 43.1 43.3 42.7 42.3 44.7 44.3 42.8 43.9 40.8 42.5 41.4 43.0 43.5 41.4 43.2 44.8 44.4 42.3 42.3 42.4 0.4

4.084 4.011 4.030 4.272 4.048 3.783 3.891 4.076 4.342 3.889 3.499 3.607 3.747 3.668 3.449 3.643 3.688 3.642 3.747 3.051 4.032 3.275 4.048 4.187 4.223 4.194 3.087 3.084 3.396 4.515 2.900 3.778 0.250

z

H- high, L- low based on testing in 2004., P- parent.

The original classification of the entries made on the basis of one year of testing in 2004 for isoflavone content remained somewhat consistent with comprehensive testing in 2005 and 2006. In Group A, for each of the 10 populations, the line originally designated as high had significantly higher isoflavone levels than the line identified as low. Within Group B, there was modest misplacement of 1
2 lines within each class for one or both populations (Table 2). Ignoring this limitation we first compared class means using only the designated genotypes without parents or check lines. In Group A, the mean isoflavone content of the entries classified as high isoflavone was significantly greater than the mean of the entries classified as low isoflavone (Table 3). In Group B, the mean isoflavone content of the high class was significantly greater from both the mean of the intermediate class and the mean of the low class, which were the same (Table 4). In Group A the mean of days to maturity and the mean of protein content of the entries classified as high isoflavone were significantly different from the means of the entries classified as low isoflavone (Table 3). In

Group B, the mean protein content of the high isoflavone class was significantly lower than the mean of the low isoflavone class (Table 4). There were no differences among class means for maturity in Group B. Since the original classification of genotypes into specific isoflavone class was not absolute, a GGE genotype-by-trait biplot was used to estimate the interrelationships between traits for each group independent of the original class structure and including all genotypes. The genotype by trait biplot explained 76% of the total variation of Group A (Fig. 1) and 77% of the total variation of Group B (Fig. 2). Vectors for each trait were drawn on the biplot originating from the origin. The cosine of the angle between the vectors of any two traits approximates the correlation co-efficient (Yan and Kang 2003). The patterns of the GGE biplots are the same in both experiments (Figs. 1 and 2). The acute angles between the vectors of isoflavone and maturity indicate that these traits are positively associated. Isoflavone content is negatively correlated with protein content, as evidenced by the large obtuse angle between the trait vectors. The 90 degree angle between the

MURPHY ET AL. * SOYBEAN ISOFLAVONE CONTENT

481

Table 2. Means and standard errors of isoflavone content, maturity, oil content, protein content and yield of 35 soybean (Glycine max L. Merr.) genotypes belonging to Group B grown in 10 environments in 2005 and 2006 Entry number 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35

Pedigree RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT Angora CK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 RCAT AngoraCK-01 Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35RCAT Angora Heinong35 RCAT Angora CK-01 Crown OAC Kent

Isoflavone classificationz

Isoflavone (mg g1)

Maturity (days)

Oil (%)

Protein (%)

Yield (mt ha1)

L L L L L I I I I I H H H H H L L L L L I I I I I H H H H H P P P C C Mean SEM

2365 2644 3100 2583 2995 2808 2599 3092 2541 2667 3476 2734 3509 2899 3460 2197 2547 2323 2296 2196 2273 3109 2047 2860 2319 2974 3086 2601 2056 3245 1576 3319 2611 1985 1990 2659 235

118 122 121 125 124 121 118 130 120 120 127 120 124 130 125 120 126 119 125 119 121 125 117 126 121 120 123 116 116 125 113 125 120 117 122 122 4

20.8 20.7 20.8 20.4 20.7 21.3 21.3 20.8 20.9 20.8 21.3 21.1 20.4 20.5 20.5 20.8 20.2 20.7 20.4 20.7 20.4 20.4 20.3 20.1 20.4 20.8 20.4 20.0 20.3 21.2 19.3 21.1 20.4 21.1 21.1 20.6 0.2

43.1 42.4 42.4 43.0 41.8 41.2 41.2 41.3 41.8 41.4 41.0 41.3 42.6 42.1 42.4 42.7 41.8 42.1 43.6 41.9 43.0 42.1 43.3 42.9 43.1 42.2 41.3 43.3 43.2 40.8 44.5 41.1 43.5 41.6 42.1 42.3 0.4

4.395 4.024 4.219 3.857 4.208 3.844 4.141 3.977 4.324 3.998 4.235 4.458 4.176 4.208 4.229 3.775 3.723 3.902 4.066 3.748 3.869 3.568 3.930 3.939 3.809 3.495 3.757 3.827 3.746 3.958 3.282 3.963 4.331 4.138 4.393 3.986 0.290

z

L, low; I, intermediate; H, high based on testing in 2004; P, parent; C, check.

isoflavone vector and the yield and oil vectors indicates that isoflavone content is not associated with these traits.

Table 3. Mean total isoflavone (mg g1), oil (%), protein (%), maturity (days), and yield (mt ha1) of 20 soybean genotypes classified as high isoflavone or low isoflavone in Group A

DISCUSSION Using a wide range of genetic material evaluated across numerous environments, this study found that total isoflavone content was negatively correlated with Table 4. Mean total isoflavone (ug g1), oil (%), protein (%), maturity (days), and yield (mt ha1) of 30 soybean genotypes classified as high, intermediate or low isoflavone in Group B

Mean Mean Trait name

High

Low

SEM

Isoflavone (mg g1) Oil (%) Protein (%) Maturity (days) Yield (mt ha1)

2843a 20.8a 42.0a 121a 3.8a

2326b 20.6a 42.5b 123b 3.9a

190 0.23 0.37 3.9 0.21

Trait name

a,b Means within a row followed by the same letter are not significantly different according to a Tukey’s multiple means comparison (a 0.01).

1

Isoflavone (mg g Oil (%) Protein (%) Maturity (days) Yield (mt ha1)

)

High

Intermediate

Low

SEM

3005a 20.7a 42.0a 123a 4.0a

2627bc 20.7a 42.1ab 121a 3.9a

2524c 20.6a 42.5b 122a 4.0a

230 0.20 0.31 3.8 0.28

a-c Means within a row followed by the same letter are not significantly different according to a Tukey’s multiple means compar-

482 CANADIAN JOURNAL OF PLANT SCIENCE

Fig. 1. Vector view of the genotype-by-trait biplot, showing the interrelationships among protein content, oil content, total isoflavone content, yield and maturity for 31 soybean genotypes (Group A) grown in 10 environments in 2005 and 2005.

protein content and positively correlated with days to maturity. There was no correlation between total isoflavone content and yield. The consistent lack of an association between isoflavone levels and yield strongly suggests that the development of agronomically acceptable cultivars with widely disparate levels of seed isoflavones would be possible. The imposed population structure of the two groups based on 2004 genotypic isoflavone levels supports, with some exceptions, the results of the GGE biplot analysis. Although the environment and G E interaction were reported as significant factors for isoflavone content, the means

comparison found that differences in isoflavone content were significant between the classes. The findings that isoflavone content and protein content are negatively correlated are consistent with the results of other studies (Chiari et al. 2004; Seguin et al. 2004; Primomo et al. 2005; Al-Tawaha and Seguin 2006). Although the relationship between isoflavone and protein was negative, genotypes were identified, such as entry 8 in Group A and entries 13 and 15 in Group B that had high isoflavone content (approximately 3500 mg g1) and moderate protein levels (42%). Class comparison of means showed that high isoflavone entries had

Fig. 2. Vector view of the genotype-by-trait biplot, showing interrelationships among protein content, oil content, total isoflavone content, yield and maturity for 35 soybean genotypes (Group B) grown in 10 environments in 2005 and 2006.

MURPHY ET AL. * SOYBEAN ISOFLAVONE CONTENT

significantly lower protein content than low isoflavone entries. Based on these results, the potential for developing high isoflavone/mid-protein soybean cultivars exists; however, the development of cultivars with high levels of both compounds may be challenging. The GGE biplot demonstrated a positive association between maturity and isoflavone content, consistent with Primomo et al. (2005) and Wang et al. (2000). Longer season genotypes, those that mature later in the season, have increased isoflavone content. Several authors have suggested that delayed seeding date increases isoflavone content (Al-Tawaha and Seguin 2006; Aussenac et al. 1998; Tsukamoto et al. 1995). This relationship is most likely a function of the relationship between temperature during seed fill and isoflavone accumulation. Cooler temperatures during the seed fill period increase isoflavone content (Caldwell et al. 2005; Lozovaya et al. 2005; Murphy 2007). The positive relationship between isoflavone content and maturity may be related to the timing of seed filling in later maturing lines, which occurs later in the season when temperatures can be expected to usually be lower in the Canadian soybean production regions. While the means comparison across classes did not support the positive correlation between isoflavone and maturity identified through GGE analysis these results may be due to the inexact nature of the original classification system. Selection within a cross was made only on the basis of isoflavone level among a relatively limited number of genotypes which did result in some wide maturity differences between classes within some of the populations. A class-cross means comparison in Group B found that within the RCAT Angora CK01 cross, the days to maturity of the high isoflavone entries were significantly later than the days to maturity of the low and intermediate entries. In the RCAT Angora  Heinong 35 cross, there was no significant difference in days to maturity across the classes (data not shown). CONCLUSIONS In a breeding program designed to develop either high or low isoflavone soybean cultivars, knowledge of the interrelationships among key agronomic or seed traits is important. This study, utilizing a large number of genotypes from a number of diverse crosses evaluated across a wide range of environments, provides a good base to study these associations. Although environment will influence the absolute expression of seed isoflavone levels the results of this study did not identify any associations between isoflavone levels and seed or agronomic traits that would impede the development of either high or low isoflavone genotypes for the major soybean growing regions of Canada. ACKNOWLEDGEMENTS Research was supported by the Ontario Soybean Growers and the Ontario Ministry of Agriculture,

483

Food and Rural Affairs. The author thanks Doug Jessop and Dale Anderson for lab analyses and Bryan Stirling and Dennis Fischer for expert technical assistance with field trials. Also, thanks to Dr. Duane Falk for biplot analysis. Ablett, G. R. and Tanner, J. W. 1993. RCAT Angora soybean. Can. J. Plant Sci. 73: 179
180. Al-Tawaha, A. M. and Seguin, P. 2006. Seeding date, row spacing, and weed effects on soybean isoflavone concentrations and other seed characteristics. Can. J. Plant Sci. 86: 1079
1087. AOAC International, 2003. Official Method 2001.10: Determination of isoflavones in soy and selected foods containing soy. Extraction, saponification and liquid chromatography. Official Methods of Analysis of AOAC International, Ed. W. Horowitz, Ch. 45, pp. 96
100. Aussenac, T., Lacombe, S. and Dayde, J. 1998. Quantification of isoflavones by capillary zone electrophoresis in soybean seeds: Effects of variety and environment. Am. J Clin. Nutr. 68: 1480S
1485. Caldwell, C. R., Britz, S. J. and Mirecki, R. M. 2005. Effect of temperature, elevated carbon dioxide, and drought during seed development on the isoflavone content of dwarf soybean (Glycine max (L.) Merrill) grown in controlled environments. J. Agric. Food Chem. 53: 1125
1129. Charron, C. S., Allen, F. L., Johnson, R. D., Pantalone, V. R. and Sams, C. E. 2005. Correlations of oil and protein with isoflavone concentration in soybean (Glycine max (L.) Merr.). J. Agric. Food Chem. 53: 7128
7135. Chiari, L., Piovesan, N. D., Naoe, L. K., Jose, I. C., Viana, J. M. S., Moreira, M. A. and deBarros, E. G. 2004. Genetic parameters relating isoflavone and protein content in soybean seeds. Euphytica 138: 55
60. Cober, E. R. and D Voldeng, H. 2000. Developing high-protein, high-yield soybean populations and lines. Crop Sci. 40: 39
42. Fehr, W. R., Caviness, C. E., Burmood, D. T. and Pennington, J. S. 1971. Stage of development descriptions for soybeans, Glycine max (L.) Merrill. Crop Sci. 11: 929
931. Kosslak, R. M., Brookland, R., Barkei, J., Paaren, H. E. and Appelbaum, E. R. 1987. Induction of Bradyrhizobium japonicum common NOD genes by isoflavones isolated from Glycine max. Proc. Natl. Acad. Sci. USA 84: 7428
7432. Lozovaya, V. V., Lygin, A. V., Ulanov, A. V., Nelson, R. L., Dayde, J. and Widholm, J. M. 2005. Effect of temperature and soil moisture status during seed development on soybean seed isoflavone concentration and composition. Crop Sci. 45: 1934
1940. Lund, R. E. 1975. Tables for an approximate test for outliers in linear models. Technometrics 17: 473
476. Morrison, M. J., Cober, E. R., Saleem, M. F., McLaughlin, N. B., Fre´geau-Reid, J., Ma, B. L., and Woodrow, L. 2008. Changes in isoflavone concentration with 58 years of genetic improvement of short-season soybean cultivars in Canada. Crop Sci. 48: 2201
2208. Murphy, S. E. 2007. Evaluation of isoflavone content in soybean: Genotype by environment interaction and association with other seed traits. M.S. thesis. Univ of Guelph, Guelph, Ontario. Primomo, V. S., Poysa, V., Ablett, G. R., Jackson, C. J. and Rajcan, I. 2005. Agronomic performance of recombinant inbred line populations segregating for isoflavone content in soybean seeds. Crop Sci. 45: 2203
2211.

484 CANADIAN JOURNAL OF PLANT SCIENCE Rivera-Vargas, L., Schmitthenner, A. F. and Graham, T. L. 1993. Soybean flavonoid effects on and metabolism by Phytopthora sojae. Phytochemistry 32: 851
857. SAS Institute, Inc. 2003. SAS for Windows. 9.1.3. SAS Institute Inc., Cary, NC. Seguin, P., Zheng, W., Smith, D. L. and Deng, W. 2004. Isoflavone content of soybean cultivars grown in eastern Canada. J. Sci. Food. Agric. 84: 1327
1332. Setchell, K. D. 1998. Phytoestrogens: The biochemistry, physiology, and implications for human health of soy isoflavones. Am. J. Clin. Nutr 68(6 Suppl.): 1333S
1346S. Tsukamoto, C., Shimada, S., Igita, K., Kudou, S., Kokubun, M., Okubo, K. and Kitamura, K. 1995. Factors affecting isoflavone content in soybean seeds: Changes in isoflavones, saponins,

and composition of fatty acids at different temperatures during seed development. J. Agric. Food Chem. 43: 1184
1192. Wang, C., Sherrard, M., Pagadala, S., Wixon, R. and Scott, R. A. 2000. Isoflavone content among maturity group 0 to II soybeans. J. Am. Oil Chem. Soc. 77: 483
487. Yan, W. 2001. GGEbiplot
A windows application for graphical analysis of multienvironment trial data and other types of two-way data. Agron. J. 93: 1111
1118. Yan, W. and Kang, M. S. 2003. GGE biplot analysis: A graphical tool for breeders, geneticists, and agronomists. CRC Press LLC, Boca Raton, FL. Yin, X. and Vyn, T. J. 2005. Relationships of isoflavone, oil, and protein in seed with yield of soybean. Agron. J. 97: 1314
1321.