Workflow for the Quantification of Soluble and Insoluble Carbohydrates in Soybean Seed.

Soybean seed composition has a profound impact on its market value and commercial use as an important commodity. Increases in oil and protein content have been historically pursued by breeders and genetic engineers; consequently, rapid methods for their quantification are well established. The interest in complete carbohydrate profiles in mature seeds, on the other hand, has recently increased due to numerous attempts to redirect carbohydrates into oil and protein or to offer specialty seed with a specific sugar profile to meet animal nutritional requirements. In this work, a sequential protocol for quantifying reserve and structural carbohydrates in soybean seed was developed and validated. Through this procedure, the concentrations of soluble sugars, sugar alcohols, starch, hemicellulose, and crystalline cellulose can be determined in successive steps from the same starting material using colorimetric assays, LC-MS/MS, and GC-MS. The entire workflow was evaluated using internal standards to estimate the recovery efficiency. Finally, it was successfully applied to eight soybean genotypes harvested from two locations, and the resulting correlations of carbohydrate and oil or protein are presented. This methodology has the potential not only to guide soybean cultivar optimization processes but also to be expanded to other crops with only slight modifications.

Effectiveness of a Seed Plate Assay for Evaluating Charcoal Rot Resistance in Soybean and the Relationship to Field Performance.

Charcoal rot of soybean, caused by Macrophomina phaseolina, is a disease of economic significance in the United States. Although there are soybean cultivars with moderate resistance, identifying and quantifying resistance is challenging. Existing assays are time consuming, and results are often highly variable. The objectives of this research were to (i) create a reproducible seed plate assay (SPA) for charcoal rot resistance and (ii) correlate field-based disease assessments with SPA results on diverse soybean accessions. To develop the SPA, surface-disinfected seeds from eight soybean genotypes (representing three susceptible and five resistant cultivars) were placed on water agar plates inoculated with M. phaseolina. After incubation at room temperature in darkness for 7 days, percent germination was determined for each cultivar relative to the germination on noninoculated plates. Results from SPA were in general agreement with published responses. None of the soybean genotypes showed complete resistance to M. phaseolina. For the second objective, charcoal rot resistance in 18 soybean accessions was assayed with SPA, and results were analyzed for correlation with field disease assessments from Stuttgart, AR, from 2011 to 2014 and from Rohwer, AR, in 2011 and 2012. SPA consistently categorized soybean genotype resistance compared with field disease assessment averages, and results were consistent with previously published resistance determinations. SPA was significantly correlated with percent height of internal stem discoloration (PHSD) at Stuttgart from 2011 to 2013 and in 2012 at Rohwer, with root and stem severity (RSS) at Rohwer in 2012, and with tap root colonization (CFU) at Stuttgart in 2012. SPA was significantly correlated to yield at Stuttgart in 2011, 2013, and 2014, and in 2011 and 2012 at Rohwer. Yield was not correlated to RSS, PHSD, or CFU at either location or in any year. Therefore, SPA is a reproducible and rapid assay for charcoal rot resistance in soybean and is significantly associated to field performance.

Across-environment seed protein stability and genetic architecture of seed components in soybean.

The recent surge in the plant-based protein market has resulted in high demands for soybean genotypes with improved grain yield, seed protein and oil content, and essential amino acids (EAAs). Given the quantitative nature of these traits, complex interactions among seed components, as well as between seed components and environmental factors and management practices, add complexity to the development of desired genotypes. In this study, the across-environment seed protein stability of 449 genetically diverse plant introductions was assessed, revealing that genotypes may display varying sensitivities to such environmental stimuli. The EAAs valine, phenylalanine, and threonine showed the highest variable importance toward the variation in stability, while both seed protein and oil contents were among the explanatory variables with the lowest importance. In addition, 56 single nucleotide polymorphism (SNP) markers were significantly associated with various seed components. Despite the strong phenotypic Pearson's correlation observed among most seed components, many independent genomic regions associated with one or few seed components were identified. These findings provide insights for improving the seed concentration of specific EAAs and reducing the negative correlation between seed protein and oil contents.

Deciphering genetic factors contributing to enhanced resistance against Cercospora leaf blight in soybean (Glycine max L.) using GWAS analysis.

Cercospora leaf blight (CLB), caused by Cercospora cf. flagellaris, C. kikuchii, and C. cf. sigesbeckiae, is a significant soybean [Glycine max (L.) Merr.] disease in regions with hot and humid conditions causing yield loss in the United States and Canada. There is limited information regarding resistant soybean cultivars, and there have been marginal efforts to identify the genomic regions underlying resistance to CLB. A Genome-Wide Association Study was conducted using a diverse panel of 460 soybean accessions from maturity groups III to VII to identify the genomic regions associated to the CLB disease. These accessions were evaluated for CLB in different regions of the southeastern United States over 3 years. In total, the study identified 99 Single Nucleotide Polymorphism (SNPs) associated with the disease severity and 85 SNPs associated with disease incidence. Across multiple environments, 47 disease severity SNPs and 23 incidence SNPs were common. Candidate genes within 10 kb of these SNPs were involved in biotic and abiotic stress pathways. This information will contribute to the development of resistant soybean germplasm. Further research is warranted to study the effect of pyramiding desirable genomic regions and investigate the role of identified genes in soybean CLB resistance.

Genomic analysis and characterization of new loci associated with seed protein and oil content in soybeans.

Breeding for increased protein without a reduction in oil content in soybeans [Glycine max (L.) Merr.] is a challenge for soybean breeders but an expected goal. Many efforts have been made to develop new soybean varieties with high yield in combination with desirable protein and/or oil traits. An elite line, R05-1415, was reported to be high yielding, high protein, and low oil. Several significant quantitative trait loci (QTL) for protein and oil were reported in this line, but many of them were unstable across environments or genetic backgrounds. Thus, a new study under multiple field environments using the Infinium BARCSoySNP6K BeadChips was conducted to detect and confirm stable genomic loci for these traits. Genetic analyses consistently detected a single major genomic locus conveying these two traits with remarkably high phenotypic variation explained (R2 ), varying between 24.2% and 43.5%. This new genomic locus is located between 25.0 and 26.7 Mb, distant from the previously reported QTL and did not overlap with other commonly reported QTL and the recently cloned gene Glyma.20G085100. Homolog analysis indicated that this QTL did not result from the paracentric chromosome inversion with an adjacent genomic fragment that harbors the reported QTL. The pleiotropic effect of this QTL could be a challenge for improving protein and oil simultaneously; however, a further study of four candidate genes with significant expressions in the seed developmental stages coupled with haplotype analysis may be able to pinpoint causative genes. The functionality and roles of these genes can be determined and characterized, which lay a solid foundation for the improvement of protein and oil content in soybeans.

Physical and textural properties of functional edible protein films from soybean using an innovative 3D printing technology.

Increasing market demand for sustainable, environmentally friendly edible film materials has called for the development of new customizable production methods utilizing emerging technologies such as 3D printing. We hereby report a new method to generate functional edible soy protein isolate films prepared from three types of soybeans (AR-R11-7999, MO-S17-17168, and MO-S17-19874R) using an innovative 3D printing technology. The protein contents in AR-R11-7999, MO-S17-17168, and MO-S17-19874R soybean meals and their corresponding protein isolates were 40.0, 39.1, and 39.9; and 84.5, 84.7, and 87.3 % (w/w, dry basis), respectively. Response surface methodology was used to maximize the tensile and puncture strength and minimize the thickness of the 3D-printed edible films using protein concentration, plasticizer concentration (glycerol), and drying time as the independent variables. The optimized film production conditions were determined as soy protein concentration: 8.91%, plasticizer concentration: 3.00%, and drying time: 3.98 h with a desirability value of 0.7428. The optimized conditions were then successfully verified with the original soybean lot with a nonsignificant difference in physical properties. At the optimized conditions, the 3D-printed edible films using three soybean lots revealed: 0.108-0.114 mm thickness; 14.79-16.07 MPa tensile strength; 6.97-8.20 N puncture strength; 90.81-91.53, -1.89 to -1.31, and 14.85-17.25 were color parameters L*, a*, and b*, respectively; 1.22-1.36 g/cm3 density; and 104.4-105.7% elongation at break ratio (%). PRACTICAL APPLICATION: Edible soy protein films produced by an extrusion-based 3D printing approach are highly customizable and precise, and could be produced at an industrial scale. This newly produced environment-friendly soy protein-based edible film can serve as an alternate packaging to synthetic plastics and reduce the environmental landfill problem while adding value to soybean produced in the mid-south United States.

Genome-wide association analysis of sucrose and alanine contents in edamame beans.

The sucrose and Alanine (Ala) content in edamame beans significantly impacts the sweetness flavor of edamame-derived products as an important attribute to consumers' acceptance. Unlike grain-type soybeans, edamame beans are harvested as fresh beans at the R6 to R7 growth stages when beans are filled 80-90% of the pod capacity. The genetic basis of sucrose and Ala contents in fresh edamame beans may differ from those in dry seeds. To date, there is no report on the genetic basis of sucrose and Ala contents in the edamame beans. In this study, a genome-wide association study was conducted to identify single nucleotide polymorphisms (SNPs) related to sucrose and Ala levels in edamame beans using an association mapping panel of 189 edamame accessions genotyped with a SoySNP50K BeadChip. A total of 43 and 25 SNPs was associated with sucrose content and Ala content in the edamame beans, respectively. Four genes (Glyma.10g270800, Glyma.08g137500, Glyma.10g268500, and Glyma.18g193600) with known effects on the process of sucrose biosynthesis and 37 novel sucrose-related genes were characterized. Three genes (Gm17g070500, Glyma.14g201100 and Glyma.18g269600) with likely relevant effects in regulating Ala content and 22 novel Ala-related genes were identified. In addition, by summarizing the phenotypic data of edamame beans from three locations in two years, three PI accessions (PI 532469, PI 243551, and PI 407748) were selected as the high sucrose and high Ala parental lines for the perspective breeding of sweet edamame varieties. Thus, the beneficial alleles, candidate genes, and selected PI accessions identified in this study will be fundamental to develop edamame varieties with improved consumers' acceptance, and eventually promote edamame production as a specialty crop in the United States.

Effect of Planting Date and Cultivar Maturity in Edamame Quality and Harvest Window.

Edamame is a food-grade soybean [Glycine max (L.) Merr.] that is harvested immature between the R6 and R7 reproductive stages. To be labeled as a premium product, the edamame market demands large pod size and intense green color. A staggered harvest season is critical for the commercial industry to post-harvest process the crop in a timely manner. Currently, there is little information to assist in predicting the optimum time to harvest edamame when the pods are at their collective largest size and greenest color. The objectives of this study were to assess the impact of cultivar, planting date, and harvest date on edamame color, pod weight, and a newly minted Edamame Harvest Quality Index combining both aforementioned factors. And to predict edamame harvest quality based on phenological stages, thermal units, and planting dates. We observed that pod color and weight depended on the cultivar, planting date, and harvest date combination. Our results also indicated that edamame quality is increased with delayed planting dates and that quality was dependent on harvest date with a quadratic negative response to delaying harvest. Maximum quality depended on cultivar and planting and harvest dates, but it remained stable for an interval of 18-27 days around the peak. Finally, we observed that the number of days between R1 and harvest was consistently identified as a key factor driving edamame quality by both stepwise regression and neural network analysis. These research results will help define a planting and harvest strategy for edamame production in Arkansas and the United States Mid-South.