Integrative omics analysis elucidates the genetic basis underlying seed weight and oil content in soybean.

Synergistic optimization of key agronomic traits by traditional breeding has dramatically enhanced crop productivity in the past decades. However, the genetic basis underlying coordinated regulation of yield- and quality-related traits remains poorly understood. Here, we dissected the genetic architectures of seed weight and oil content by combining genome-wide association studies (GWAS) and transcriptome-wide association studies (TWAS) using 421 soybean (Glycine max) accessions. We identified 26 and 33 genetic loci significantly associated with seed weight and oil content by GWAS, respectively, and detected 5,276 expression quantitative trait loci (eQTLs) regulating expression of 3,347 genes based on population transcriptomes. Interestingly, a gene module (IC79), regulated by two eQTL hotspots, exhibited significant correlation with both seed weigh and oil content. Twenty-two candidate causal genes for seed traits were further prioritized by TWAS, including Regulator of Weight and Oil of Seed 1 (GmRWOS1), which encodes a sodium pump protein. GmRWOS1 was verified to pleiotropically regulate seed weight and oil content by gene knockout and overexpression. Notably, allelic variations of GmRWOS1 were strongly selected during domestication of soybean. This study uncovers the genetic basis and network underlying regulation of seed weight and oil content in soybean and provides a valuable resource for improving soybean yield and quality by molecular breeding.

Mutations in the genes responsible for the synthesis of furan fatty acids resolve the light-induced off-odor in soybean oil.

Soybean oil is the second most produced edible vegetable oil and is used for many edible and industrial materials. Unfortunately, it has the disadvantage of 'reversion flavor' under photooxidative conditions, which produces an off-odor and decreases the quality of edible oil. Reversion flavor and off-odor are caused by minor fatty acids in the triacylglycerol of soybean oil known as furan fatty acids, which produce 3-methyl-2,4-nonanedione (3-MND) upon photooxidation. As a solution to this problem, a reduction in furan fatty acids leads to a decrease in 3-MND, resulting in a reduction in the off-odor induced by light exposure. However, there are no reports on the genes related to the biosynthesis of furan fatty acids in soybean oil. In this study, four mutant lines showing low or no furan fatty acid levels in soybean seeds were isolated from a soybean mutant library. Positional cloning experiments and homology search analysis identified two genes responsible for furan fatty acid biosynthesis in soybean: Glyma.20G201400 and Glyma.04G054100. Ectopic expression of both genes produced furan fatty acids in transgenic soybean hairy roots. The structure of these genes is different from that of the furan fatty acid biosynthetic genes in photosynthetic bacteria. Homologs of these two group of genes are widely conserved in the plant kingdom. The purified oil from the furan fatty acid mutant lines had lower amounts of 3-MND and reduced off-odor after light exposure, compared with oil from the wild-type.

Bioengineering of Soybean Oil and Its Impact on Agronomic Traits.

Soybean is a major oil crop and is also a dominant source of nutritional protein. The 20% seed oil content (SOC) of soybean is much lower than that in most oil crops and the fatty acid composition of its native oil cannot meet the specifications for some applications in the food and industrial sectors. Considerable effort has been expended on soybean bioengineering to tailor fatty acid profiles and improve SOC. Although significant advancements have been made, such as the creation of high-oleic acid soybean oil and high-SOC soybean, those genetic modifications have some negative impacts on soybean production, for instance, impaired germination or low protein content. In this review, we focus on recent advances in the bioengineering of soybean oil and its effects on agronomic traits.

QTL Mapping of Palmitic Acid Content Using Specific-Locus Amplified Fragment Sequencing (SLAF-Seq) Genotyping in Soybeans (Glycine max L.).

Soybeans are essential crops that supply protein and oil. The composition and contents of soybean fatty acids are relevant to human health and have a significant relationship with soybean oil processing and applications. Identifying quantitative trait locus (QTL) genes related to palmitic acid could facilitate the development of a range of nutritive soybean cultivars using molecular marker-assisted selection. In this study, we used a cultivar with higher palmitic acid content, 'Dongnong42', and a lower palmitic acid content cultivar, 'Hobbit', to establish F2:6 recombinant inbred lines. A high-density genetic map containing 9980 SLAF markers was constructed and distributed across 20 soybean chromosomes. The genetic map contained a total genetic distance of 2602.58 cM and an average genetic distance of 0.39 cM between adjacent markers. Two QTLs related to palmitic acid content were mapped using inclusive composite interval mapping, explaining 4.2-10.1% of the phenotypic variance in three different years and environments, including the QTL included in seed palmitic 7-3, which was validated by developing SSR markers. Based on the SNP/Indel and significant differential expression analyses of Dongnong42 and Hobbit, two genes, Glyma.15g119700 and Glyma.15g119800, were selected as candidate genes. The high-density genetic map, QTLs, and molecular markers will be helpful for the map-based cloning of palmitic acid content genes. These could be used to accelerate breeding for high nutritive value cultivars via molecular marker-assisted breeding.

Overexpression of Soybean GmWRI1a Stably Increases the Seed Oil Content in Soybean.

WRINKLED1 (WRI1), an APETALA2/ethylene-responsive-element-binding protein (AP2/EREBP) subfamily transcription factor, plays a crucial role in the transcriptional regulation of plant fatty acid biosynthesis. In this study, GmWRI1a was overexpressed in the soybean cultivar 'Dongnong 50' using Agrobacterium-mediated transformation to generate three transgenic lines with high seed oil contents. PCR and Southern blotting analysis showed that the T-DNA was inserted into the genome at precise insertion sites and was stably inherited by the progeny. Expression analysis using qRT-PCR and Western blotting indicated that GmWRI1a and bar driven by the CaMV 35S promoter were significantly upregulated in the transgenic plants at different developmental stages. Transcriptome sequencing results showed there were obvious differences in gene expression between transgenic line and transgenic receptor during seed developmental stages. KEGG analysis found that the differentially expressed genes mainly annotated to metabolic pathways, such as carbohydrated metabolism and lipid metabolism. A 2-year single-location field trial revealed that three transgenic lines overexpressing GmWRI1a (GmWRI1a-OE) showed a stable increase in seed oil content of 4.97-10.35%. Importantly, no significant effect on protein content and yield was observed. Overexpression of GmWRI1a changed the fatty acid composition by increasing the linoleic acid (C18:2) content and decreasing the palmitic acid (C16:0) content in the seed. The three GmWRI1a-OE lines showed no significant changes in agronomic traits. The results demonstrated that the three GmWRI1a overexpression lines exhibited consistent increases in seed oil content compared with that of the wild type and did not significantly affect the seed yield and agronomic traits. The genetic engineering of GmWRI1a will be an effective strategy for the improvement of seed oil content and value in soybean.

Integrating omics approaches to discover and prioritize candidate genes involved in oil biosynthesis in soybean.

Soybean is a major source of edible protein and oil. Oil content is a quantitative trait that is significantly determined by genetic and environmental factors. Over the past 30 years, a large volume of soybean genetic, genomic, and transcriptomic data have been accumulated. Nevertheless, integrative analyses of such data remain scarce, in spite of their importance for crop improvement. We hypothesized that the co-occurrence of genomic regions for oil-related traits in different studies may reveal more stable regions encompassing important genetic determinants of oil content and quality in soybean. We integrated publicly available data, obtained with distinct techniques, to discover and prioritize candidate genes involved in oil biosynthesis and regulation in soybean. We detected key fatty acid biosynthesis genes (e.g., BCCP2 and ACCase, FADs, KAS family proteins) and several transcription factors, which are likely regulators of oil biosynthesis. In addition, we identified new candidates for seed oil accumulation and quality, such as Glyma.03G213300 and Glyma.19G160700, which encode a translocator protein homolog and a histone acetyltransferase, respectively. Further, oil and protein genomic hotspots are strongly associated with breeding and not with domestication, suggesting that soybean domestication prioritized other traits. The genes identified here are promising targets for breeding programs and for the development of soybean lines with increased oil content and quality.

Quantitative proteomic and lipidomics analyses of high oil content GmDGAT1-2 transgenic soybean illustrate the regulatory mechanism of lipoxygenase and oleosin.

KEY MESSAGE: Proteomic and lipidomics analyses of WT and GmDGAT1-2 transgenic soybeans showed that GmDGAT1-2 over-expression induced lipoxygenase down-regulatation and oleoin up-regulatation, which significantly changed the compositions and total fatty acid. The main goal of soybean breeding is to increase the oil content. Diacylglycerol acyltransferase (DGAT) is a key rate-limiting enzyme in fatty acid metabolism and may regulate oil content. Herein, 10 GmDGAT genes were isolated from soybean and transferred into wild-type (WT) Arabidopsis. The total fatty acid was 1.2 times higher in T3 GmDGAT1-2 transgenic Arabidopsis seeds than in WT. Therefore, GmDGAT1-2 was transferred into WT soybean (JACK), and four T3 transgenic soybean lines were obtained. The results of high-performance gas chromatography and Soxhlet extractor showed that, compared with those of JACK, oleic acid (18:1), and total fatty acid levels in transgenic soybean plants were much higher, but linoleic acid (18:2) was lower than WT. Palmitic acid (16:0), stearic acid (18:0), and linolenic acid (18:3) were not significantly different. For mechanistic studies, 436 differentially expressed proteins (DEPs) and 180 differentially expressed metabolites (DEMs) were identified between WT (JACK) and transgenic soybean pods using proteomic and lipidomics analyses. Four lipoxygenase proteins were down-regulated in linoleic acid metabolism while four oleosin proteins were up-regulated in the final oil formation. The results showed an increase in the total fatty acid and 18:1 composition, and a decrease in the 18:2 composition of fatty acid. Our study brings new insights into soybean genetic transformation and the deep study of molecular mechanism that changes the total fatty acid, 18:1, and 18:2 compositions in GmDGAT1-2 transgenic soybean.

Circular RNAs acting as ceRNAs mediated by miRNAs may be involved in the synthesis of soybean fatty acids.

Soybean oil is composed of fatty acids and glycerol. The content and composition of fatty acids partly determine the quality of soybean seeds. Circular RNAs (circRNAs) are endogenous non-coding RNAs that competitively bind to microRNAs (miRNAs) through miRNA recognition elements, thereby acting as sponges to regulate the expression of target genes. Although circRNAs have been identified previously in soybean, only their expression has been investigated without exploration of the competitive endogenous RNAs (ceRNAs) network of circRNAs-miRNAs-mRNAs. In this study, circRNAs in immature pods of a low linolenic acid soybean Mutant 72' (MT72) and the wild-type control 'Jinong 18' (JN18) were systematically identified and analyzed at 30 and 40 days after flowering using high-throughput sequencing technology. We identified 6377 circRNAs, of which 114 were differentially expressed. Gene ontology and KEGG pathway analyses of targeted mRNAs in the ceRNAs network indicated that the differentially expressed circRNAs may be involved in fatty acid transport, suggesting that circRNAs may play a post-transcriptional regulatory role in soybean oil synthesis. This study provides a foundation for future exploration of the function of circRNAs in soybean and presents novel insights to guide further studies of plant circRNAs.

CRISPR/Cas9-Mediated Knockout of GmFATB1 Significantly Reduced the Amount of Saturated Fatty Acids in Soybean Seeds.

Soybean (Glycine max) oil is one of the most widely used vegetable oils across the world. Breeding of soybean to reduce the saturated fatty acid (FA) content, which is linked to cardiovascular disease, would be of great significance for nutritional improvement. Acyl-acyl carrier protein thioesterases (FATs) can release free FAs and acyl-ACP, which ultimately affects the FA profile. In this study, we identified a pair of soybean FATB coding genes, GmFATB1a and GmFATB1b. Mutants that knock out either or both of the GmFATB1 genes were obtained via CRISPR/Cas9. Single mutants, fatb1a and fatb1b, showed a decrease in leaf palmitic and stearic acid contents, ranging from 11% to 21%. The double mutant, fatb1a:1b, had a 42% and 35% decrease in palmitic and stearic acid content, displayed growth defects, and were male sterility. Analysis of the seed oil profile revealed that fatb1a and fatb1b had significant lower palmitic and stearic acid contents, 39-53% and 17-37%, respectively, while that of the unsaturated FAs were the same. The relative content of the beneficial FA, linoleic acid, was increased by 1.3-3.6%. The oil profile changes in these mutants were confirmed for four generations. Overall, our data illustrate that GmFATB1 knockout mutants have great potential in improving the soybean oil quality for human health.

Genome-wide association study identifies candidate genes related to oleic acid content in soybean seeds.

BACKGROUND: Soybean oil is a complex mixture of five fatty acids (palmitic, stearic, oleic, linoleic, and linolenic). Soybean oil with a high oleic acid content is desirable because this monounsaturated fatty acid improves the oxidative stability of the oil. To investigate the genetic architecture of oleic acid in soybean seeds, 260 soybean germplasms from Northeast China were collected as natural populations. A genome-wide association study (GWAS) was conducted on a panel of 260 germplasm resources. RESULTS: Phenotypic identification results showed that the oleic acid content varied from 8.2 to 35.0%. A total of 2,311,337 single-nucleotide polymorphism (SNP) markers were obtained. GWAS analysis showed that there were many genes related to oleic acid content with a contribution rate of 7%. The candidate genes Glyma.11G229600.1 on chromosome 11 and Glyma.04G102900.1 on chromosome 4 were detected in a 2-year-long GWAS. The candidate gene Glyma.11G229600.1 showed a positive correlation with the oleic acid content, and the correlation coefficient was 0.980, while Glyma.04G102900.1 showed a negative correlation, with a coefficient of - 0.964. CONCLUSIONS: Glyma.04G102900.1 on chromosome 4 and Glyma.11G229600.1 on chromosome 11 were detected in both analyses (2018 and 2019). Glyma.04G102900.1 and Glyma.11G229600.1 are new key candidate genes related to oleic acid in soybean seeds. These results will be useful for high-oleic soybean breeding.

Stearidonic-Enriched Soybean Oil Modulates Obesity, Glucose Metabolism, and Fatty Acid Profiles Independently of Akkermansia muciniphila.

SCOPE: Previous studies have suggested that diets rich in omega-3 and low in omega-6 long-chain polyunsaturated fatty acids (PUFAs) can limit the development of metabolic syndrome (MetS). Transgenic soybeans yielding oils enriched for omega-3 PUFAs represent a new and readily-available option for incorporating omega-3 PUFAs into diets to provide health benefits. METHODS AND RESULTS: Transgenic soybean oils, enriched for either stearidonic acid (SDA) or eicosapentaenoic acid (EPA), are incorporated into diets to test their effects on limiting the development of MetS in a mouse model of diet-induced obesity. Supplementation with SDA- but not EPA-enriched oils improved features of MetS compared to feeding a control wild-type oil. Because previous studies have linked the gut microorganism Akkermansia muciniphila to the metabolic effects of feeding omega-3 PUFAs, the causal contribution of A. muciniphila to mediating the metabolic benefits provided by SDA-enriched diets is investigated. Although A. muciniphila is not required for SDA-induced metabolic improvements, this microorganism does modulate levels of saturated and mono-unsaturated fatty acids in host adipose tissues. CONCLUSION: Together, these findings support the utilization of SDA-enriched diets to modulate weight gain, glucose metabolism, and fatty acid profiles of liver and adipose tissue.

Three-dimensional genetic networks among seed oil-related traits, metabolites and genes reveal the genetic foundations of oil synthesis in soybean.

Although the biochemical and genetic basis of lipid metabolism is clear in Arabidopsis, there is limited information concerning the relevant genes in Glycine max (soybean). To address this issue, we constructed three-dimensional genetic networks using six seed oil-related traits, 52 lipid metabolism-related metabolites and 54 294 SNPs in 286 soybean accessions in total. As a result, 284 and 279 candidate genes were found to be significantly associated with seed oil-related traits and metabolites by phenotypic and metabolic genome-wide association studies and multi-omics analyses, respectively. Using minimax concave penalty (MCP) and smoothly clipped absolute deviation (SCAD) analyses, six seed oil-related traits were found to be significantly related to 31 metabolites. Among the above candidate genes, 36 genes were found to be associated with oil synthesis (27 genes), amino acid synthesis (four genes) and the tricarboxylic acid (TCA) cycle (five genes), and four genes (GmFATB1a, GmPDAT, GmPLDα1 and GmDAGAT1) are already known to be related to oil synthesis. Using this information, 133 three-dimensional genetic networks were constructed, 24 of which are known, e.g. pyruvate-GmPDAT-GmFATA2-oil content. Using these networks, GmPDAT, GmAGT and GmACP4 reveal the genetic relationships between pyruvate and the three major nutrients, and GmPDAT, GmZF351 and GmPgs1 reveal the genetic relationships between amino acids and seed oil content. In addition, GmCds1, along with average temperature in July and the rainfall from June to September, influence seed oil content across years. This study provides a new approach for the construction of three-dimensional genetic networks and reveals new information for soybean seed oil improvement and the identification of gene function.

Quantitative trait loci analysis of seed oil content and composition of wild and cultivated soybean.

BACKGROUND: Soybean oil is a major source of edible oil, and the domestication of wild soybean has resulted in significant changes in oil content and composition. Extensive efforts have been made to identify genetic loci that are related to soybean oil traits. The objective of this study was to identify quantitative trait loci (QTLs) related to soybean seed oil and compare the fatty acid composition between wild and cultivated soybean. RESULTS: Using the specific-locus amplified fragment sequencing (SLAF-seq) method, a total of 181 recombinant inbred lines (RILs) derived from a cross between wild soybean ZYD00463 (Glycine soja) and cultivated soybean WDD01514 (Glycine max) were genotyped. Finally, a high-density genetic linkage map comprising 11,398 single-nucleotide polymorphism (SNP) markers on 20 linkage groups (LGs) was constructed. Twenty-four stable QTLs for seed oil content and composition were identified by model-based composite interval mapping (CIM) across multiple environments. Among these QTLs, 23 overlapped with or were adjacent to previously reported QTLs. One QTL, qPA10_1 (5.94-9.98 Mb) on Chr. Ten is a novel locus for palmitic acid. In the intervals of stable QTLs, some interesting genes involved in lipid metabolism were detected. CONCLUSIONS: We developed 181 RILs from a cross between wild soybean ZYD00463 and cultivated soybean WDD01514 and constructed a high-density genetic map using the SLAF-seq method. We identified 24 stable QTLs for seed oil content and compositions, which includes qPA10_1 on Chr. 10, a novel locus for palmitic acid. Some interesting genes in the QTL regions were also detected. Our study will provide useful information for scientists to learn about genetic variations in lipid metabolism between wild and cultivated soybean.

Metabolic engineering of soybean seeds for enhanced vitamin E tocochromanol content and effects on oil antioxidant properties in polyunsaturated fatty acid-rich germplasm.

Soybean seeds produce oil enriched in oxidatively unstable polyunsaturated fatty acids (PUFAs) and are also a potential biotechnological platform for synthesis of oils with nutritional omega-3 PUFAs. In this study, we engineered soybeans for seed-specific expression of a barley homogentisate geranylgeranyl transferase (HGGT) transgene alone and with a soybean γ-tocopherol methyltransferase (γ-TMT) transgene. Seeds for HGGT-expressing lines had 8- to 10-fold increases in total vitamin E tocochromanols, principally as tocotrienols, with little effect on seed oil or protein concentrations. Tocochromanols were primarily in δ- and γ-forms, which were shifted largely to α- and ß-tocochromanols with γ-TMT co-expression. We tested whether oxidative stability of conventional or PUFA-enhanced soybean oil could be improved by metabolic engineering for increased vitamin E antioxidants. Selected lines were crossed with a stearidonic acid (SDA, 18:4Δ6,9,12,15)-producing line, resulting in progeny with oil enriched in SDA and α- or γ-linoleic acid (ALA, 18:3Δ9,12,15 or GLA, 18:3Δ6,9,12), from transgene segregation. Oil extracted from HGGT-expressing lines had ≥6-fold increase in free radical scavenging activity compared to controls. However, the oxidative stability index of oil from vitamin E-enhanced lines was ~15% lower than that of oil from non-engineered seeds and nearly the same or modestly increased in oil from the GLA, ALA and SDA backgrounds relative to controls. These findings show that soybean is an effective platform for producing high levels of free-radical scavenging vitamin E antioxidants, but this trait may have negative effects on oxidative stability of conventional oil or only modest improvement of the oxidative stability of PUFA-enhanced oil.

Genome-wide association mapping for seed protein and oil contents using a large panel of soybean accessions.

Soybean is globally cultivated primarily for its protein and oil. The protein and oil contents of the seeds are quantitatively inherited traits determined by the interaction of numerous genes. In order to gain a better understanding of the molecular foundation of soybean protein and oil content for the marker-assisted selection (MAS) of high quality traits, a population of 185 soybean germplasms was evaluated to identify the quantitative trait loci (QTLs) associated with the seed protein and oil contents. Using specific length amplified fragment sequencing (SLAF-seq) technology, a total of 12,072 single nucleotide polymorphisms (SNPs) with a minor allele frequency (MAF) ≥ 0.05 were detected across the 20 chromosomes (Chr), with a marker density of 78.7 kbp. A total of 31 SNPs located on 12 of the 20 soybean chromosomes were correlated with seed protein and oil content. Of the 31 SNPs that were associated with the two target traits, 31 beneficial alleles were identified. Two SNP markers, namely rs15774585 and rs15783346 on Chr 07, were determined to be related to seed oil content both in 2015 and 2016. Three SNP markers, rs53140888 on Chr 01, rs19485676 on Chr 13, and rs24787338 on Chr 20 were correlated with seed protein content both in 2015 and 2016. These beneficial alleles may potentially contribute towards the MAS of favorable soybean protein and oil characteristics.

Meta-analysis and transcriptome profiling reveal hub genes for soybean seed storage composition during seed development.

Soybean is an important crop providing edible oil and protein source. Soybean oil and protein contents are quantitatively inherited and significantly affected by environmental factors. In this study, meta-analysis was conducted based on soybean physical maps to integrate quantitative trait loci (QTLs) from multiple experiments in different environments. Meta-QTLs for seed oil, fatty acid composition, and protein were identified. Of them, 11 meta-QTLs were located on hot regions for both seed oil and protein. Next, we selected 4 chromosome segment substitution lines with different seed oil and protein contents to characterize their 3 years of phenotype selection in the field. Using strand-specific RNA-sequencing analysis, we profile the time-course transcriptome patterns of soybean seeds at early maturity, middle maturity, and dry seed stages. Pairwise comparison and K-means clustering analysis revealed 7,482 differentially expressed genes and 45 expression patterns clusters. Weighted gene coexpression network analysis uncovered 46 modules of gene expression patterns. The 2 most significant coexpression networks were visualized, and 7 hub genes were identified that were involved in soybean oil and seed storage protein accumulation processes. Our results provided a transcriptome dataset for soybean seed development, and the candidate hub genes represent a foundation for further research.

Improved oil quality in transgenic soybean seeds by RNAi-mediated knockdown of GmFAD2-1B.

Soybean oil contains approximately 20% oleic acid and 63% polyunsaturated fatty acids, which limits its uses in food products and industrial applications because of its poor oxidative stability. Increasing the oleic acid content in soybean seeds provides improved oxidative stability and is also beneficial to human health. Endoplasmic reticulum-associated delta-12 fatty acid desaturase 2 (FAD2) is the key enzyme responsible for converting oleic acid (18:1) precursors to linoleic acid (18:2) in the lipid biosynthetic pathway. In this study, a 390-bp conserved sequence of GmFAD2-1B was used to trigger a fragment of RNAi-mediated gene knockdown, and a seed-specific promoter of the ß-conglycinin alpha subunit gene was employed to downregulate the expression of this gene in soybean seeds to increase the oleic acid content. PCR and Southern blot analysis showed that the T-DNA had inserted into the soybean genome and was stably inherited by the progeny. In addition, the expression analysis indicated that GmFAD2-1B was significantly downregulated in the seeds by RNAi-mediated post-transcription gene knockdown driven by the seed-specific promoter. The oleic acid content significantly increased from 20 to ~ 80% in the transgenic seeds, and the linoleic and linolenic acid content decreased concomitantly in the transgenic lines compared with that in the wild types. The fatty acid profiles also exhibited steady changes in three consecutive generations. However, the total protein and oil contents and agronomic traits of the transgenic lines did not show a significant difference compared with the wild types.

Genotyping-by-Sequencing-Based Investigation of the Genetic Architecture Responsible for a ∼Sevenfold Increase in Soybean Seed Stearic Acid.

Soybean oil is highly unsaturated but oxidatively unstable, rendering it nonideal for food applications. Until recently, the majority of soybean oil underwent partial chemical hydrogenation, which produces trans fats as an unavoidable consequence. Dietary intake of trans fats and most saturated fats are conclusively linked to negative impacts on cholesterol levels and cardiovascular health. Two major soybean oil breeding targets are: (1) to reduce or eliminate the need for chemical hydrogenation, and (2) to replace the functional properties of partially hydrogenated soybean oil. One potential solution is the elevation of seed stearic acid, a saturated fat which has no negative impacts on cardiovascular health, from 3 to 4% in typical cultivars to > 20% of the seed oil. We performed QTL analysis of a population developed by crossing two mutant lines, one with a missense mutation affecting a stearoyl-acyl-carrier protein desaturase gene resulting in ∼11% seed stearic acid crossed to another mutant, A6, which has 24-28% seed stearic acid. Genotyping-by-sequencing (GBS)-based QTL mapping identified 21 minor and major effect QTL for six seed oil related traits and plant height. The inheritance of a large genomic deletion affecting chromosome 14 is the basis for largest effect QTL, resulting in ∼18% seed stearic acid. This deletion contains SACPD-C and another gene(s); loss of both genes boosts seed stearic acid levels to ≥ 18%. Unfortunately, this genomic deletion has been shown in previous studies to be inextricably correlated with reduced seed yield. Our results will help inform and guide ongoing breeding efforts to improve soybean oil oxidative stability.

Direct stacking of sequence-specific nuclease-induced mutations to produce high oleic and low linolenic soybean oil.

BACKGROUND: The ability to modulate levels of individual fatty acids within soybean oil has potential to increase shelf-life and frying stability and to improve nutritional characteristics. Commodity soybean oil contains high levels of polyunsaturated linoleic and linolenic acid, which contribute to oxidative instability - a problem that has been addressed through partial hydrogenation. However, partial hydrogenation increases levels of trans-fatty acids, which have been associated with cardiovascular disease. Previously, we generated soybean lines with knockout mutations within fatty acid desaturase 2-1A (FAD2-1A) and FAD2-1B genes, resulting in oil with increased levels of monounsaturated oleic acid (18:1) and decreased levels of linoleic (18:2) and linolenic acid (18:3). Here, we stack mutations within FAD2-1A and FAD2-1B with mutations in fatty acid desaturase 3A (FAD3A) to further decrease levels of linolenic acid. Mutations were introduced into FAD3A by directly delivering TALENs into fad2-1a fad2-1b soybean plants. RESULTS: Oil from fad2-1a fad2-1b fad3a plants had significantly lower levels of linolenic acid (2.5 %), as compared to fad2-1a fad2-1b plants (4.7 %). Furthermore, oil had significantly lower levels of linoleic acid (2.7 % compared to 5.1 %) and significantly higher levels of oleic acid (82.2 % compared to 77.5 %). Transgene-free fad2-1a fad2-1b fad3a soybean lines were identified. CONCLUSIONS: The methods presented here provide an efficient means for using sequence-specific nucleases to stack quality traits in soybean. The resulting product comprised oleic acid levels above 80 % and linoleic and linolenic acid levels below 3 %.

Landscape of genomic diversity and trait discovery in soybean.

Cultivated soybean [Glycine max (L.) Merr.] is a primary source of vegetable oil and protein. We report a landscape analysis of genome-wide genetic variation and an association study of major domestication and agronomic traits in soybean. A total of 106 soybean genomes representing wild, landraces, and elite lines were re-sequenced at an average of 17x depth with a 97.5% coverage. Over 10 million high-quality SNPs were discovered, and 35.34% of these have not been previously reported. Additionally, 159 putative domestication sweeps were identified, which includes 54.34 Mbp (4.9%) and 4,414 genes; 146 regions were involved in artificial selection during domestication. A genome-wide association study of major traits including oil and protein content, salinity, and domestication traits resulted in the discovery of novel alleles. Genomic information from this study provides a valuable resource for understanding soybean genome structure and evolution, and can also facilitate trait dissection leading to sequencing-based molecular breeding.