Natural variation of domestication-related genes contributed to latitudinal expansion and adaptation in soybean.

Soybean is a major source of protein and edible oil worldwide. Originating from the Huang-Huai-Hai region, which has a temperate climate, soybean has adapted to a wide latitudinal gradient across China. However, the genetic mechanisms responsible for the widespread latitudinal adaptation in soybean, as well as the genetic basis, adaptive differentiation, and evolutionary implications of theses natural alleles, are currently lacking in comprehensive understanding. In this study, we examined the genetic variations of fourteen major gene loci controlling flowering and maturity in 103 wild species, 1048 landraces, and 1747 cultivated species. We found that E1, E3, FT2a, J, Tof11, Tof16, and Tof18 were favoured during soybean improvement and selection, which explained 75.5% of the flowering time phenotypic variation. These genetic variation was significantly associated with differences in latitude via the LFMM algorithm. Haplotype network and geographic distribution analysis suggested that gene combinations were associated with flowering time diversity contributed to the expansion of soybean, with more HapA clustering together when soybean moved to latitudes beyond 35°N. The geographical evolution model was developed to accurately predict the suitable planting zone for soybean varieties. Collectively, by integrating knowledge from genomics and haplotype classification, it was revealed that distinct gene combinations improve the adaptation of cultivated soybeans to different latitudes. This study provides insight into the genetic basis underlying the environmental adaptation of soybean accessions, which could contribute to a better understanding of the domestication history of soybean and facilitate soybean climate-smart molecular breeding for various environments.

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 quantitative trait loci and candidate genes for seed folate content in soybean.

KEY MESSAGE: From 61 QTL mapped, a stable QTL cluster of 992 kb was discovered on chromosome 5 for folate content and a putative candidate gene, Glyma.05G237500, was identified. Folate (vitamin B9) is one of the most essential micronutrients whose deficiencies lead to various health defects in humans. Herein, we mapped the quantitative trait loci (QTL) underlying seed folate content in soybean using recombinant inbred lines developed from cultivars, ZH35 and ZH13, across four environments. We identified 61 QTL on 12 chromosomes through composite interval mapping, with phenotypic variance values ranging from 1.68 to 24.68%. A major-effect QTL cluster (qFo-05) was found on chromosome 5, spanning 992 kb and containing 134 genes. Through gene annotation and single-locus haplotyping analysis of qFo-05 in a natural soybean population, we identified seven candidate genes significantly associated with 5MTHF and total folate content in multiple environments. RNA-seq analysis showed a unique expression pattern of a hemerythrin RING zinc finger gene, Glyma.05G237500, between both parental cultivars during seed development, which suggest the gene might regulate folate content in soybean. This is the first study to investigate QTL underlying folate content in soybean and provides new insight for molecular breeding to improve folate content in soybean.

Effect of Origin, Seed Coat Color, and Maturity Group on Seed Isoflavones in Diverse Soybean Germplasm.

Soybeans are grown worldwide owing to their protein, oil, and beneficial bioactive compounds. Genetic and environmental factors influence soybean seed isoflavones. In the present study, we profiled the seed isoflavones in world diverse soybean germplasm grown in two locations over two years in China. Significant differences (p < 0.001) were observed between the accessions, accession origins, seed coat colors, and maturity groups for individual and total isoflavone (TIF) content. TIF content of the soybean accessions ranged from 677.25 µg g-1 to 5823.29 µg g-1, representing an 8-fold difference. USA soybean accessions showed the highest mean TIF content (3263.07 µg g-1), followed by Japan (2521.26 µg g-1). Soybean with black seed coat showed the highest (3236.08 µg g-1) TIF concentration. Furthermore, isoflavone levels were significantly higher in late-maturity groups. Correlation analysis revealed significant positive associations between individual and TIF content. Malonyldaidzin and malonylgenistin showed higher correlations with TIF content (r = 0.92 and r = 0.94, respectively). The soybean accessions identified as having high and stable TIF content can be utilized in the food and pharmaceutical industries and breeding programs to develop soybean varieties with enhanced isoflavone content.

Identification of hub genes regulating isoflavone accumulation in soybean seeds via GWAS and WGCNA approaches.

Introduction: Isoflavones are the secondary metabolites synthesized by the phenylpropanoid biosynthesis pathway in soybean that benefits human and plant health. Methods: In this study, we have profiled seed isoflavone content by HPLC in 1551 soybean accessions grown in Beijing and Hainan for two consecutive years (2017 and 2018) and in Anhui for one year (2017). Results: A broad range of phenotypic variations was observed for individual and total isoflavone (TIF) content. The TIF content ranged from 677.25 to 5823.29 µg g-1 in the soybean natural population. Using a genome-wide association study (GWAS) based on 6,149,599 single nucleotide polymorphisms (SNPs), we identified 11,704 SNPs significantly associated with isoflavone contents; 75% of them were located within previously reported QTL regions for isoflavone. Two significant regions on chromosomes 5 and 11 were associated with TIF and malonylglycitin across more than 3 environments. Furthermore, the WGCNA identified eight key modules: black, blue, brown, green, magenta, pink, purple, and turquoise. Of the eight co-expressed modules, brown (r = 0.68***), magenta (r = 0.64***), and green (r = 0.51**) showed a significant positive association with TIF, as well as with individual isoflavone contents. By combining the gene significance, functional annotation, and enrichment analysis information, four hub genes Glyma.11G108100, Glyma.11G107100, Glyma.11G106900, and Glyma.11G109100 encoding, basic-leucine zipper (bZIP) transcription factor, MYB4 transcription factor, early responsive to dehydration, and PLATZ transcription factor respectively were identified in brown and green modules. The allelic variation in Glyma.11G108100 significantly influenced individual and TIF accumulation. Discussion: The present study demonstrated that the GWAS approach, combined with WGCNA, could efficiently identify isoflavone candidate genes in the natural soybean population.

Identification of Genomic Loci and Candidate Genes Related to Seed Tocopherol Content in Soybean.

Soybean seeds are primary sources of natural tocopherols used by the food and pharmaceutical industries, owing to their beneficial impacts on human health. Selection for higher tocopherol contents in seeds along with other desirable traits is an important goal in soybean breeding. In order to identify the genomic loci and candidate genes controlling tocopherol content in soybean seeds, the bulked-segregant analysis technique was performed using a natural population of soybean consisting of 1525 accessions. We constructed the bulked-segregant analysis based on 98 soybean accessions that showed extreme phenotypic variation for the target trait, consisting of 49 accessions with extremely-high and 49 accessions with extremely-low tocopherol content. A total of 144 variant sites and 109 predicted genes related to tocopherol content were identified, in which a total of 83 genes were annotated by the gene ontology functions. Furthermore, 13 enriched terms (p < 0.05) were detected, with four of them found to be highly enriched: response to lipid, response to abscisic acid, transition metal ion transmembrane transporter activity, and double-stranded DNA binding. Especially, six candidate genes were detected at 41.8−41.9 Mb genomic hotspots on chromosome 5 based on ANNOtate VARiation analysis. Among the genes, only Glyma.05G243400 carried a non-synonymous mutation that encodes a "translation elongation factor EF1A or initiation factor IF2gamma family protein" was identified. The haplotype analysis confirmed that Glyma.05G243400 exhibited highly significant variations in terms of tocopherol content across multiple experimental locations, suggesting that it can be the key candidate gene regulating soybean seed tocopherols. The present findings provide novel gene resources related to seed tocopherols for further validation by genome editing, functional characterization, and genetic improvement targeting enhanced tocopherol composition in soybean molecular breeding.

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.

The nutritional composition of the vegetable soybean (maodou) and its potential in combatting malnutrition.

Introduction: Global malnutrition continues to be a canker owing to poor eating habits and over-reliance on the major staple crops. Vegetable soybean (maodou) is gaining popularity globally as an affordable snack and vegetable. Methods: In this study, we profiled the nutritional composition of 12 soybean cultivars at the vegetable (R6-R7) and mature (R8) stages. We also conducted an RNA-seq analysis during seed development, focusing on key biosynthesis enzymes for quality traits. Results: The results showed that 100 g of maodou contained 66.54% moisture, 13.49% protein, 7.81% fatty acids, 2.47% soluble sugar, abundant content of minerals, and micronutrients, including folate (462.27 µg FW) and carotenoids (3,935.41 µg FW). Also, the isoflavone content of maodou ranged between 129.26 and 2,359.35 µg/g FW. With regard to the recommended daily allowance, 100 g fresh weight of maodou can contribute 26.98, 115.57, and 11.60% of protein, folate, and zinc, respectively, and significant proportions of other nutrients including linoleic acid (21.16%), linolenic acid (42.96%), zinc (11.60%), and iron (18.01%). On a dry weight basis, maodou has two to six folds higher contents of folate, tocopherol, and carotenoid than the mature soybean. Furthermore, RNA-seq analysis revealed that key biosynthesis enzymes of quality traits are differentially expressed during seed development and may contribute to variations in the content of quality traits at the vegetable and mature stages. Correlation analysis of quality traits at both stages revealed that protein only correlated positively with zinc at the vegetable stage but negatively correlated with total tocopherol and total fatty acid at the mature stage. Complex associations among folates, soluble sugar, and isoflavones were also identified. Discussion: This study provides insight into the nutritional contents of vegetable soybean and demonstrates that maodou is essential for meeting the nutritional requirements of most countries.

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.

Evaluation of two wild castor (Ricinus communis L.) accessions for cadmium tolerance in relation to antioxidant systems and lipid peroxidation.

The present study was conducted to assess the effect of toxicity of cadmium (Cd) on growth, tolerance index (TI), antioxidant activities, and malondialdehyde (MDA) content in two contrasting wild castor accessions (16-024 and S2-4) via hydroponic experiment (0 and 100 mg/L Cd). The results showed that Cd significantly reduced the growth rate, seedling height, root length, and shoot length of the castor accessions compared to the control, with the Cd effect being more severe in S2-4 than in 16-024. In addition, biomass response including the root and shoot fresh weight and root dry weight decreased in both accessions compared to the control. Compared to the control group, the shoot dry weight of accession S2-4 declined by 21.7%, whereas there was no change in 16-024, suggesting a level of tolerance in 16-024. Analysis of TI on all the growth parameters and biomass content revealed that accession 16-024 was highly tolerant to Cd stress than S2-4. The results further revealed that the expression of the antioxidant enzymes, viz., superoxide dismutase (SOD), catalase (CAT), non-enzymatic antioxidant, glutathione, and MDA content, was influenced by genotype. S2-4 exhibited a higher antioxidant activity (SOD, CAT) and lipid peroxidation activity than 16-024, indicative of oxidative damage from Cd stress.

SRAP analysis of the genetic diversity of wild castor (Ricinus communis L.) in South China.

Castor bean is an important seed oil crop. Castor oil is a highly demanded oil for several industrial uses. Currently, castor bean varieties suffer from low productivity and high risk of insect pests and diseases. It is in urgent need to mine elite genes from wild materials for castor breeding. 29 pairs of polymorphic SRAP primers out of 361 pairs were used to analyse the genetic diversity of 473 wild castor materials from South China. 203 bands were amplified by the 29 pairs of primers, of which 169 bands were polymorphic, with a polymorphic percentage of 83.25%. With an average number of alleles per locus (Ap) of 1.801, average number of effective alleles per locus (Ae) of 1.713 and average percentage of polymorphic loci (P) of 90.04%, these primers were proven to be useful and effective. Nei' genetic distance between the materials ranged from 1.04 to 25.02, with an average of 13.03. At the genetic distance of 25.02, the materials clustered into two major groups, consistent with the result of population structure analysis. However, more subgroups existed between 5.21 and 13.32. Although not all the materials from the same region were clustered in the same group, an obvious trend existed where the groups were related to regions to a great extent. Based on multiple indices, the genetic diversity of materials from Hainan was the lowest. However, there was not much difference between West Guangdong and Guangxi, although the former was slightly higher. Moderate genetic differentiation was observed in wild materials in South China. The genetic differentiation mainly occurred within population, with maximum differentiation in Guangxi, followed by West Guangdong and the minimum in Hainan. Nonetheless, there was an extensive geneflow between populations. The above results provided a direction for the conservation and breeding application of these materials.