A Review of Bioactive Compound Effects from Primary Legume Protein Sources in Human and Animal Health.

The global demand for sustainable and nutritious food sources has catalyzed interest in legumes, known for their rich repertoire of health-promoting compounds. This review delves into the diverse array of bioactive peptides, protein subunits, isoflavones, antinutritional factors, and saponins found in the primary legume protein sources-soybeans, peas, chickpeas, and mung beans. The current state of research on these compounds is critically evaluated, with an emphasis on the potential health benefits, ranging from antioxidant and anticancer properties to the management of chronic diseases such as diabetes and hypertension. The extensively studied soybean is highlighted and the relatively unexplored potential of other legumes is also included, pointing to a significant, underutilized resource for developing health-enhancing foods. The review advocates for future interdisciplinary research to further unravel the mechanisms of action of these bioactive compounds and to explore their synergistic effects. The ultimate goal is to leverage the full spectrum of benefits offered by legumes, not only to advance human health but also to contribute to the sustainability of food systems. By providing a comprehensive overview of the nutraceutical potential of legumes, this manuscript sets a foundation for future investigations aimed at optimizing the use of legumes in the global pursuit of health and nutritional security.

Soybean genetics, genomics, and breeding for improving nutritional value and reducing antinutritional traits in food and feed.

Soybean [Glycine max (L.) Merr.] is a globally important crop due to its valuable seed composition, versatile feed, food, and industrial end-uses, and consistent genetic gain. Successful genetic gain in soybean has led to widespread adaptation and increased value for producers, processors, and consumers. Specific focus on the nutritional quality of soybean seed composition for food and feed has further elucidated genetic knowledge and bolstered breeding progress. Seed components are historical and current targets for soybean breeders seeking to improve nutritional quality of soybean. This article reviews genetic and genomic foundations for improvement of nutritionally important traits, such as protein and amino acids, oil and fatty acids, carbohydrates, and specific food-grade considerations; discusses the application of advanced breeding technology such as CRISPR/Cas9 in creating seed composition variations; and provides future directions and breeding recommendations regarding soybean seed composition traits.

Determining Genetic Markers and Seed Compositions Related to High Test Weight in Glycine max.

Test weight, one of the primary indicators of soybean seed quality, is measured as the amount of soybean seeds in kilograms that can fit into one hectoliter. The price that growers receive for their soybean is dependent on test weight. Over the past 50 years, growers have observed a decreasing trend in test weight. Therefore, it is imperative to understand better the relationship between soybean test weight and other traits to enable breeders to select parental lines with high test weights in breeding programs to ensure the grower's profitability. The objectives of the study were to identify genetic markers associated with high test weight in soybean and to determine the correlation between high test weight and five important seed composition traits (protein, oil, sucrose, raffinose, and stachyose content). Maturity group IV and V germplasms from the USDA soybean germplasm collection were grown in Blacksburg and Warsaw in Virginia from 2019 to 2021 and were measured for all of the above traits. Results show that test weight values ranged from 62-77 kg/hL over the three years. Multiple single-nucleotide polymorphisms (SNPs) significantly associated with high test weight were found on chromosome (Chr.) 15 along with a couple on chromosome 14, and 11 candidate genes were found near these SNPs. Test weight was found to be significantly negatively correlated with oil content, inconsistently correlated with protein content in all environments, and negatively correlated but not significantly with all three sugars except for raffinose in Blacksburg 2019. We concluded that the genes that underlie test weight might be on chromosome 15, and the validated associated SNPs might be used to assist breeding selection of test weight. Breeders should pay special attention to test weight while selecting for high oil content in soybean due to their negative correlation.

Development of new mutant alleles and markers for KTI1 and KTI3 via CRISPR/Cas9-mediated mutagenesis to reduce trypsin inhibitor content and activity in soybean seeds.

The digestibility of soybean meal can be severely impacted by trypsin inhibitor (TI), one of the most abundant anti-nutritional factors present in soybean seeds. TI can restrain the function of trypsin, a critical enzyme that breaks down proteins in the digestive tract. Soybean accessions with low TI content have been identified. However, it is challenging to breed the low TI trait into elite cultivars due to a lack of molecular markers associated with low TI traits. We identified Kunitz trypsin inhibitor 1 (KTI1, Gm01g095000) and KTI3 (Gm08g341500) as two seed-specific TI genes. Mutant kti1 and kti3 alleles carrying small deletions or insertions within the gene open reading frames were created in the soybean cultivar Glycine max cv. Williams 82 (WM82) using the CRISPR/Cas9-mediated genome editing approach. The KTI content and TI activity both remarkably reduced in kti1/3 mutants compared to the WM82 seeds. There was no significant difference in terms of plant growth or maturity days of kti1/3 transgenic and WM82 plants in greenhouse condition. We further identified a T1 line, #5-26, that carried double homozygous kti1/3 mutant alleles, but not the Cas9 transgene. Based on the sequences of kti1/3 mutant alleles in #5-26, we developed markers to co-select for these mutant alleles by using a gel-electrophoresis-free method. The kti1/3 mutant soybean line and associated selection markers will assist in accelerating the introduction of low TI trait into elite soybean cultivars in the future.

Evaluate the guide RNA effectiveness via Agrobacterium-mediated transient assays in Nicotiana benthamiana.

CRISPR/Cas9-based genome editing system is a powerful tool for plant genetic improvement. However, the variable efficiency of guide RNA(s) (gRNA) represents a key limiting factor that hampers the broad application of the CRISPR/Cas9 system in crop improvement. Here, we employed the Agrobacterium-mediated transient assays to evaluate the effectiveness of gRNAs for editing genes in Nicotiana benthamiana and soybean. We designed a facile screening system based on indels that can be introduced by CRISPR/Cas9-mediated gene editing. A gRNA binding sequence (23 nucleotides) was inserted into the open reading frame of yellow fluorescent protein (YFP) gene (gRNA-YFP), which disrupted the YFP reading frame and results in no fluorescent signal when it was expressed in plant cells. Transiently co-expression of Cas9 and a gRNA targeting the gRNA-YFP gene in plant cells could restore the YFP reading frame and recover the YFP signals. We evaluated five gRNAs targeting Nicotiana benthamiana and soybean genes and confirmed the reliability of the gRNA screening system. The effective gRNAs targeting NbEDS1, NbWRKY70, GmKTI1, and GmKTI3 had been used to generate transgenic plants and resulted in expected mutations on each gene. While a gRNA targeting NbNDR1 was confirmed to be ineffective in transient assays. This gRNA indeed failed to trigger target gene mutations in stable transgenic plants. Thus, this new transient assay system can be used to validate the effectiveness of gRNAs before generating stable transgenic plants.

Genome-Wide Association Study and Genomic Selection for Proteinogenic Methionine in Soybean Seeds.

Soybean [Glycine max (L.) Merr.] seeds have an amino acid profile that provides excellent viability as a food and feed protein source. However, low concentrations of an essential amino acid, methionine, limit the nutritional utility of soybean protein. The objectives of this study were to identify genomic associations and evaluate the potential for genomic selection (GS) for methionine content in soybean seeds. We performed a genome-wide association study (GWAS) that utilized 311 soybean accessions from maturity groups IV and V grown in three locations in 2018 and 2019. A total of 35,570 single nucleotide polymorphisms (SNPs) were used to identify genomic associations with proteinogenic methionine content that was quantified by high-performance liquid chromatography (HPLC). Across four environments, 23 novel SNPs were identified as being associated with methionine content. The strongest associations were found on chromosomes 3 (ss715586112, ss715586120, ss715586126, ss715586203, and ss715586204), 8 (ss715599541 and ss715599547) and 16 (ss715625009). Several gene models were recognized within proximity to these SNPs, such as a leucine-rich repeat protein kinase and a serine/threonine protein kinase. Identification of these linked SNPs should help soybean breeders to improve protein quality in soybean seeds. GS was evaluated using k-fold cross validation within each environment with two SNP sets, the complete 35,570 set and a subset of 248 SNPs determined to be associated with methionine through GWAS. Average prediction accuracy (r 2) was highest using the SNP subset ranging from 0.45 to 0.62, which was a significant improvement from the complete set accuracy that ranged from 0.03 to 0.27. This indicated that GS utilizing a significant subset of SNPs may be a viable tool for soybean breeders seeking to improve methionine content.