Dual-function C2H2-type zinc-finger transcription factor GmZFP7 contributes to isoflavone accumulation in soybean.

Isoflavones are a class of secondary metabolites produced by legumes and play important roles in human health and plant stress tolerance. The C2H2 zinc-finger transcription factor (TF) functions in plant stress tolerance, but little is known about its function in isoflavone regulation in soybean (Glycine max). Here, we report a C2H2 zinc-finger TF gene, GmZFP7, which regulates isoflavone accumulation in soybean. Overexpressing GmZFP7 increased the isoflavone concentration in both transgenic hairy roots and plants. By contrast, silencing GmZFP7 expression significantly reduced isoflavone levels. Metabolomic and qRT-PCR analysis revealed that GmZFP7 can increase the flux of the phenylpropanoid pathway. Furthermore, dual-luciferase and electrophoretic mobility shift assays showed that GmZFP7 regulates isoflavone accumulation by influencing the expression of Isoflavone synthase 2 (GmIFS2) and Flavanone 3 ß-hydroxylase 1 (GmF3H1). In this study, we demonstrate that GmZFP7 contributes to isoflavone accumulation by regulating the expression of the gateway enzymes (GmIFS2 and GmF3H1) of competing phenylpropanoid pathway branches to direct the metabolic flux into isoflavone. A haplotype analysis indicated that important natural variations were present in GmZFP7 promoters, with P-Hap1 and P-Hap3 being the elite haplotypes. Our findings provide insight into how GmZFP7 regulates the phenylpropanoid pathway and enhances soybean isoflavone content.

Identification of genes for seed isoflavones based on bulk segregant analysis sequencing in soybean natural population.

KEY MESSAGE: We identified four hub genes for isoflavone biosynthesis based on BSA-seq and WGCNA methods and validated that GmIE3-1 positively contribute to isoflavone accumulation in soybean. Soybean isoflavones are secondary metabolites of great interest owing to their beneficial impact on human health. Herein, we profiled the seed isoflavone content by HPLC in 1551 soybean accessions grown in two locations for two years and constructed two extreme pools with high (4065.1 µg g-1) and low (1427.23 µg g-1) isoflavone contents to identify candidate genes involved in isoflavone biosynthesis pathways using bulk segregant analysis sequencing (BSA-seq) approach. The results showed that the average sequencing depths were 50.3× and 65.7× in high and low pools, respectively. A total of 23,626 polymorphic SNPs and 5299 InDels were detected between both pools and 1492 genes with different variations were identified. Based on differential genes in BSA-seq and weighted gene co-expression network analysis (WGCNA), four hub genes, Glyma.06G290400 (designated as GmIE3-1), Glyma.01G239200, Glyma.01G241500, Glyma.13G256100 were identified, encoding E3 ubiquitin-protein ligase, arm repeat protein interacting with ABF2, zinc metallopeptidase EGY3, and dynamin-related protein 3A, respectively. The allelic variation in GmIE3-1 showed a significant influence on isoflavone accumulation. The virus-induced gene silencing (VIGS) and RNAi hairy root transformation of GmIE3-1 revealed partial suppression of this gene could cause a significant decrease (P < 0.0001) of total isoflavone content, suggesting GmIE3-1 is a positive regulator for isoflavones. The present study demonstrated that the BSA-seq approach combined with WGCNA, VIGS and hairy root transformation can efficiently identify isoflavone candidate genes in soybean natural population.

The GmSNAP11 Contributes to Resistance to Soybean Cyst Nematode Race 4 in Glycine max.

Soybean cyst nematode (SCN) has devastating effects on soybean production, making it crucial to identify genes conferring SCN resistance. Here we employed next-generation sequencing-based bulked segregant analysis (BSA) to discover genomic regions, candidate genes, and diagnostic markers for resistance to SCN race 4 (SCN4) in soybean. Phenotypic analysis revealed highly significant differences among the reactions of 145 recombinant inbred lines (RILs) to SCN4. In combination with euclidean distance (ED) and Δsingle-nucleotide polymorphism (SNP)-index analyses, we identified a genomic region on Gm11 (designated as rhg1-paralog) associated with SCN4 resistance. Overexpression and RNA interference analyzes of the two candidate genes identified in this region (GmPLAC8 and GmSNAP11) revealed that only GmSNAP11 significantly contributes to SCN4 resistance. We developed a diagnostic marker for GmSNAP11. Using this marker, together with previously developed markers for SCN-resistant loci, rhg1 and Rhg4, we evaluated the relationship between genotypes and SCN4 resistance in 145 RILs and 30 soybean accessions. The results showed that all the SCN4-resistant lines harbored all the three loci, however, some lines harboring the three loci were still susceptible to SCN4. This suggests that these three loci are necessary for the resistance to SCN4, but they alone cannot confer full resistance. The GmSNAP11 and the diagnostic markers developed could be used in genomic-assisted breeding to develop soybean varieties with increased resistance to SCN4.

Natural Variation of Seed Tocopherol Composition in Diverse World Soybean Accessions from Maturity Group 0 to VI Grown in China.

Tocopherols are natural antioxidants that increase the stability of fat-containing foods and are well known for their health benefits. To investigate the variation in seed tocopherol composition of soybeans from different origins, 493 soybean accessions from different countries (China, USA, Japan, and Russia) belonging to 7 maturity groups (MG 0-VI) were grown in 2 locations (Beijing and Hainan Provinces of China) for 2 years (2017 and 2018). The results showed that significant differences (p < 0.001) were observed among the accessions and origins for individual and total tocopherol contents. The total tocopherol content ranged from 118.92 µg g-1 to 344.02 µg g-1. Accessions from the USA had the highest average concentration of γ- and total tocopherols (152.92 and 238.21 µg g-1, respectively), whereas a higher level of α-tocopherol (12.82 µg g-1) was observed in the Russian accessions. The maturity group of the accession significantly (p < 0.001) influenced all tocopherol components, and higher levels of α-, γ-, and total tocopherols were observed in early maturing accessions, while late-maturing accessions exhibited higher levels of δ-tocopherol. The inclination of tocopherol concentrations with various MGs provided further evidence of the significance of MG in soybean breeding for seed tocopherol components. Furthermore, the correlation between the seed tocopherol components and geographical factors revealed that α-, γ-, and total tocopherols had significant positive correlations with latitude, while δ-tocopherol showed an opposite trend. The elite accessions with high and stable tocopherol concentrations determined could be used to develop functional foods, industrial materials, and breeding lines to improve tocopherol composition in soybean seeds.

Origin, Maturity Group and Seed Coat Color Influence Carotenoid and Chlorophyll Concentrations in Soybean Seeds.

Soybean (Glycine max (L.) Merrill) seeds are abundant in physiologically active metabolites, including carotenoids and chlorophylls, and are used as an affordable source of functional foods that promote and maintain human health. The distribution and variation of soybean seed metabolites are influenced by plant genetic characteristics and environmental factors. Here, we investigated the effects of germplasm origin, genotype, seed coat color and maturity group (MG) on the concentration variation of carotenoid and chlorophyll components in 408 soybean germplasm accessions collected from China, Japan, the USA and Russia. The results showed that genotype, germplasm origin, seed color, and MG were significant variation sources of carotenoid and chlorophyll contents in soybean seeds. The total carotenoids showed about a 25-fold variation among the soybean germplasms, with an overall mean of 12.04 µg g-1. Russian soybeans yielded 1.3-fold higher total carotenoids compared with Chinese and Japanese soybeans. Similarly, the total chlorophylls were substantially increased in Russian soybeans compared to the others. Soybeans with black seed coat color contained abundant concentrations of carotenoids, with mainly lutein (19.98 µg g-1), ß-carotene (0.64 µg g-1) and total carotenoids (21.04 µg g-1). Concentrations of lutein, total carotenoids and chlorophylls generally decreased in late MG soybeans. Overall, our results demonstrate that soybean is an excellent dietary source of carotenoids, which strongly depend on genetic factors, germplasm origin, MG and seed coat color. Thus, this study suggests that soybean breeders should consider these factors along with environmental factors in developing carotenoid-rich cultivars and related functional food resources.

Profiling of naturally occurring folates in a diverse soybean germplasm by HPLC-MS/MS.

Soybean is a rich source of folates. We optimised the extraction and detection of folates from soybean seeds by HPLC-MS/MS and analysed the folate content and composition of 1074 accessions. Total folate content ranged from 64.51 to 691.24 µg/100 g fresh weight, with 10-fold variation, and 60 elite accessions with over 400 µg/100 g of total folate were identified. The most abundant component was 5-CHO-H4folate, which accounted for an average of 60% of total folate content. Seed-coat colour, seed weight, ecoregion, and accession type significantly affected soybean folate content. Furthermore, 5-CH3-H4folate correlated positively with seed protein (r = 0.24***) and negatively with oil (r = -0.26***). The geographical distribution of folate according to accession origin revealed that accessions from Northeast China contain higher amounts of total folate and 5-CHO-H4folate. This study provides comprehensive and novel insights into the folate profile of soybean, which will benefit soybean breeding for folate enhancement.

Assessment of the Genetic Structure and Diversity of Soybean (Glycine max L.) Germplasm Using Diversity Array Technology and Single Nucleotide Polymorphism Markers.

Knowledge of the genetic structure and diversity of germplasm collections is crucial for sustainable genetic improvement through hybridization programs and rapid adaptation to changing breeding objectives. The objective of this study was to determine the genetic diversity and population structure of 281 International Institute of Tropical Agriculture (IITA) soybean accessions using diversity array technology (DArT) and single nucleotide polymorphism (SNP) markers for the efficient utilization of these accessions. From the results, the SNP and DArT markers were well distributed across the 20 soybean chromosomes. The cluster and principal component analyses revealed the genetic diversity among the 281 accessions by grouping them into two stratifications, a grouping that was also evident from the population structure analysis, which divided the 281 accessions into two distinct groups. The analysis of molecular variance revealed that 97% and 98% of the genetic variances using SNP and DArT markers, respectively, were within the population. Genetic diversity indices such as Shannon's diversity index, diversity and unbiased diversity revealed the diversity among the different populations of the soybean accessions. The SNP and DArT markers used provided similar information on the structure, diversity and polymorphism of the accessions, which indicates the applicability of the DArT marker in genetic diversity studies. Our study provides information about the genetic structure and diversity of the IITA soybean accessions that will allow for the efficient utilization of these accessions in soybean improvement programs, especially in Africa.

Molecular-Assisted Distinctness and Uniformity Testing Using SLAF-Sequencing Approach in Soybean.

Distinctness, uniformity and stability (DUS) testing of cultivars through morphological descriptors is an important and compulsory part of soybean breeding. Molecular markers are usually more effective and accurate in describing the genetic features for the identification and purity assessment of cultivars. In the present study, we assessed the distinctness and uniformity of five soybean cultivars using both single nucleotide polymorphism (SNP) markers developed by specific-locus amplified fragment sequencing (SLAF-seq) technology, and simple sequence repeat (SSR) markers. The phylogenetic tree and principal component analysis (PCA) from both the SLAF-seq and SSR methods showed a clear distinction among cultivars Zhonghuang 18, Zhonghuang 68 and Zhonghuang 35, while no clear distinction was observed between cultivars Zhonghuang 13 and Hedou 13. Using the SLAF-seq method, we determined the proportion of homozygous loci for the five soybean cultivars. The heterozygosity of each individual plant was estimated for the assessment of cultivar purity and the purity levels of the five soybean cultivars ranged from 91.89% to 93.96%. To further validate the applicability of the SLAF-seq approach for distinctness testing, we used the SNP information of 150 soybean cultivars with different origins. The cultivars were also distinguished clearly. Taken together, SLAF-seq can be used as an accurate and reliable method in the assessment of the distinctness and uniformity of soybean cultivars.

Seed isoflavone profiling of 1168 soybean accessions from major growing ecoregions in China.

Soybean (Glycine max L. Merrill) isoflavones are secondary metabolites of great interest because of their beneficial impact on human health. We profiled the seed isoflavone composition of 1168 soybean accessions collected from diverse ecoregions of China in three locations over two years. We observed significant differences in isoflavone content among the accessions, accession types, years of growth and ecoregions of origin. Total isoflavone (TIF) concentration of the soybean accessions ranged from 745 µg g-1 to 5253.98 µg g-1, which represents a 7-fold difference. The highest mean TIF concentration (2689.27 µg g-1) was observed in the Huang Huai Hai Valley Region (HR) accessions, followed by accessions from the Southern Region (SR) and Northern Region (NR) with TIF concentration of 2518.91 µg g-1 and 1942.78 µg g-1, respectively. Thirty-five accessions were identified as elite soybean resources based on their higher TIF concentration (4024.74 µg g-1 to 5253.98 µg g-1). Pairwise correlation analysis showed significant positive correlations between individual isoflavones and TIF concentrations. Malonyldaidzin and malonylgenistin showed the highest correlations with TIF concentration (r = 0.90 and r = 0.92, respectively), whereas acetyldaidzin showed the lowest correlation. The main isoflavone components had significant negative correlations with latitude and longitude, indicating that the geographical origin of the accessions influenced their seed isoflavone composition. Based on principal component analysis, glycosides and malonylglycosides of isoflavones were the major discriminative components for the soybean accessions. The present study demonstrated the geographical distribution of soybean seed isoflavone concentrations across the main ecoregion of China. The identified soybean accessions with both high and low TIF concentrations, which are desirable materials for industrial uses and could also be used as parents to breed soybean lines with improved isoflavone quantity and composition in the seeds.

CERES-Maize Model for Determining the Optimum Planting Dates of Early Maturing Maize Varieties in Northern Nigeria.

Field trials were carried out in the Sudan Savannah of Nigeria to assess the usefulness of CERES-maize crop model as a decision support tool for optimizing maize production through manipulation of plant dates. The calibration experiments comprised of 20 maize varieties planted during the dry and rainy seasons of 2014 and 2015 at Bayero University Kano and Audu Bako College of Agriculture Dambatta. The trials for model evaluation were conducted in 16 different farmer fields across the Sudan (Bunkure and Garun-Mallam) and Northern Guinea (Tudun-Wada and Lere) Savannas using two of the calibrated varieties under four different sowing dates. The model accurately predicted grain yield, harvest index, and biomass of both varieties with low RMSE-values (below 5% of mean), high d-index (above 0.8), and high r-square (above 0.9) for the calibration trials. The time series data (tops weight, stem and leaf dry weights) were also predicted with high accuracy (% RMSEn above 70%, d-index above 0.88). Similar results were also observed for the evaluation trials, where all variables were simulated with high accuracies. Estimation efficiencies (EF)-values above 0.8 were observed for all the evaluation parameters. Seasonal and sensitivity analyses on Typic Plinthiustalfs and Plinthic Kanhaplustults in the Sudan and Northern Guinea Savannas were conducted. Results showed that planting extra early maize varieties in late July and early maize in mid-June leads to production of highest grain yields in the Sudan Savanna. In the Northern Guinea Savanna planting extra-early maize in mid-July and early maize in late July produced the highest grain yields. Delaying planting in both Agro-ecologies until mid-August leads to lower yields. Delaying planting to mid-August led to grain yield reduction of 39.2% for extra early maize and 74.4% for early maize in the Sudan Savanna. In the Northern Guinea Savanna however, delaying planting to mid-August resulted in yield reduction of 66.9 and 94.3% for extra-early and early maize, respectively.