Identification of genes differentially expressed between developing seeds of different soybean varieties
ABSTRACT: Soybean is a rich source of protein and oil and a primary feedstock for biodiesel production. Previous research on soybean indicated that protein, oil and yield are controlled quantitatively in soybean seeds. However, genetic mechanisms controlling seed composition and yield in soybean remain unknown. We used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean varieties, which differ in seed weight, yield, protein content and oil content. Some of the differentially expressed genes identified in this study may play important roles in controlling these traits. The soybean plants of two soybean varieties Minsoy and Archer and two recombinant inbred lines from the cross that are similar in maturity but differ in yield were grown in St Paul, Minnesota during the summers of 2007 and 2008. In 2007, each line was planted as a single row. In 2008, a randomized complete block (RCB) design was used and each line had 3 replicates planted 1-2 weeks apart. Within each replicate, two rows per line were planted. Seeds were harvested at three developmental stages, namely, seed length = 2 mm, 3.5 mm, and 5-6 mm, which correspond approximately to soybean reproductive stages R4, R5 and early R6, respectively. In 2007 three independent samples were collected for each line and developmental stage. In 2008, two seed samples (one from each row) were collected for each line at each stage within each replicate. The pairs of seed samples were then pooled. Thus, three sets of independent tissue samples were collected for RNA extraction and hybridization on Affymetrix microarrys.
Project description:Soybean is a rich source of protein and oil and a primary feedstock for biodiesel production. Previous research on soybean indicated that protein, oil and yield are controlled quantitatively in soybean seeds. However, genetic mechanisms controlling seed composition and yield in soybean remain unknown. We used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean varieties, which differ in seed weight, yield, protein content and oil content. Some of the differentially expressed genes identified in this study may play important roles in controlling these traits. Overall design: The soybean plants of two soybean varieties Minsoy and Archer and two recombinant inbred lines from the cross that are similar in maturity but differ in yield were grown in St Paul, Minnesota during the summers of 2007 and 2008. In 2007, each line was planted as a single row. In 2008, a randomized complete block (RCB) design was used and each line had 3 replicates planted 1-2 weeks apart. Within each replicate, two rows per line were planted. Seeds were harvested at three developmental stages, namely, seed length = 2 mm, 3.5 mm, and 5-6 mm, which correspond approximately to soybean reproductive stages R4, R5 and early R6, respectively. In 2007 three independent samples were collected for each line and developmental stage. In 2008, two seed samples (one from each row) were collected for each line at each stage within each replicate. The pairs of seed samples were then pooled. Thus, three sets of independent tissue samples were collected for RNA extraction and hybridization on Affymetrix microarrys.
Project description:Soybean is a major source of protein and oil and a primary feedstock for biodiesel production. Research on soybean seed composition and yield has revealed that protein, oil and yield are controlled quantitatively and quantitative trait loci (QTL) have been identified for each of these traits. However, very limited information is available regarding the genetic mechanisms controlling seed composition and yield. To help address this deficiency, we used Affymetrix Soybean GeneChips® to identify genes that are differentially expressed between developing seeds of the Minsoy and Archer soybean cultivars, which differ in seed weight, yield, protein content and oil content. A total of 700 probe sets were found to be expressed at significantly different (defined as having an adjusted p-value below or equal to 0.05 and an at least 2-fold difference) levels between the two cultivars at one or more of the three developmental stages and in at least one of the two years assayed. Comparison of data from soybeans collected in two different years revealed that 97 probe sets were expressed at significantly different levels in both years. Functional annotations were assigned to 78% of these 97 probe sets based on the SoyBase Affymetrix™ GeneChip® Soybean Genome Array Annotation. Genes involved in receptor binding/activity and protein binding are overrepresented among the group of 97 probe sets that were differentially expressed in both years assayed. Probe sets involved in growth/development, signal transduction, transcription, defense/stress response and protein and lipid metabolism were also identified among the 97 probe sets and their possible implications in the regulation of agronomic traits are discussed. As the Minsoy and Archer soybean cultivars differ with respect to seed size, yield, protein content and lipid content, some of the differentially expressed probe sets identified in this study may thus play important roles in controlling these traits. Others of these probe sets may be involved in regulation of general seed development or metabolism. All microarray data and expression values after GCRMA are available at the Gene Expression Omnibus (GEO) at NCBI (http://www.ncbi.nlm.nih.gov/geo), under accession number GSE21598.
Project description:Soybean is an important oil-producing crop in the Fabaceae family and there are increasing demands for soybean oil and other soybean products. Genetic improvement of soybean is needed to increase its production. In order to provide genetic diversity and resources for identifying important genes, a new ethyl methane sulfonate (EMS) mutagenized soybean population was generated using the newly released germplasm, JTN-5203 (maturity group V). Treatment of soybean seeds with 60 mM EMS concentration was found to be suitable for inducing mutation. A total of 1,820 M1 individuals were produced from 15,000 treated seeds. The resulting M2 population was planted in the field for phenotyping. After harvest, seed traits including total oil, protein, starch, moisture content, fatty acid and amino acid compositions were measured by NIR. Phenotypic variations observed in this population include changes in leaf morphology, plant architecture, seed compositions, and yield. Of most interest, we identified plants with increased amounts of total protein (50% vs. 41% for control) and plants with higher amounts of total oil (25% vs. 21.2% control). Similarly, we identified plants with increases in oleic acid content and decreases in linoleic acid and linolenic acid. This EMS mutant population will be used for further studies including screening for various traits such as amino acid pathways, allergens, phytic acids, and other important soybean agronomic traits. In addition, these mutant individuals will be evaluated in the next generation to assess the heritability. Beneficial traits from these mutants can be exploited for future soybean breeding programs. This germplasm can also be used for discovering novel mutant alleles and for functional gene expression analysis using reverse genetics tools such as TILLING.
Project description:BACKGROUND: Soybean (Glycine max) seeds are the primary source of edible oil in the United States. Despite its widespread utility, soybean oil is oxidatively unstable. Until recently, the majority of soybean oil underwent chemical hydrogenation, a process which also generates trans fats. An alternative to chemical hydrogenation is genetic modification of seed oil through identification and introgression of mutant alleles. One target for improvement is the elevation of a saturated fat with no negative cardiovascular impacts, stearic acid, which typically constitutes a minute portion of seed oil (~3%). RESULTS: We examined radiation induced soybean mutants with moderately increased stearic acid (10-15% of seed oil, ~3-5 X the levels in wild-type soybean seeds) via comparative whole genome hybridization and genetic analysis. The deletion of one SACPD isoform encoding gene (SACPD-C) was perfectly correlated with moderate elevation of seed stearic acid content. However, SACPD-C deletion lines were also found to have altered nodule fatty acid composition and grossly altered morphology. Despite these defects, overall nodule accumulation and nitrogen fixation were unaffected, at least under laboratory conditions. CONCLUSIONS: Although no yield penalty has been reported for moderate elevated seed stearic acid content in soybean seeds, our results demonstrate that genetic alteration of seed traits can have unforeseen pleiotropic consequences. We have identified a role for fatty acid biosynthesis, and SACPD activity in particular, in the establishment and maintenance of symbiotic nitrogen fixation.
Project description:Background:Soybean oil constitutes an important source of vegetable oil and biofuel. However, high temperature and humidity adversely impacts soybean seed development, yield, and quality during plant development and after harvest. Genetic improvement of soybean tolerance to stress environments is highly desirable. Results:Transgenic soybean lines with knockdown of phospholipase D?1 (PLD?1KD) were generated to study PLD?1's effects on lipid metabolism and seed vigor under high temperature and humidity conditions. Under such stress, as compared with normal growth conditions, PLD?1KD lines showed an attenuated stress-induced deterioration during soybean seed development, which was associated with elevated expression of reactive oxygen species-scavenging genes when compared with wild-type control. The developing seeds of PLD?1KD had higher levels of unsaturation in triacylglycerol (TAG) and major membrane phospholipids, but lower levels of phosphatidic acid and lysophospholipids compared with control cultivar. Lipid metabolite and gene expression profiling indicates that the increased unsaturation on phosphatidylcholine (PC) and enhanced conversion between PC and diacylglycerol (DAG) by PC:DAG acyltransferase underlie a basis for increased TAG unsaturation in PLD?1KD seeds. Meanwhile, the turnover of PC and phosphatidylethanolamine (PE) into lysoPC and lysoPE was suppressed in PLD?1KD seeds under high temperature and humidity conditions. PLD?1KD developing seeds suffered lighter oxidative stresses than did wild-type developing seeds in the stressful environments. PLD?1KD seeds contain higher oil contents and maintained higher germination rates than the wild-type seeds. Conclusions:The study provides insights into the roles of PLD?1 in developing soybean seeds under high temperature and humidity stress. PLD?1KD decreases pre-harvest deterioration and enhances acyl editing in phospholipids and TAGs. The results indicate a way towards improving production of quality soybean seeds as foods and biofuels under increasing environmental stress.
Project description:Soybean (Glycine max) is a major contributor to the world oilseed production. Its seed oil content has been increased through soybean domestication and improvement. However, the genes underlying the selection are largely unknown. The present contribution analyzed the expression patterns of genes in the seed oil quantitative trait loci with strong selective sweep signals, then used association, functional study and population genetics to reveal a sucrose efflux transporter gene, GmSWEET39, controlling soybean seed oil content and under selection. GmSWEET39 is highly expressed in soybean seeds and encodes a plasma membrane-localized protein. Its expression level is positively correlated with soybean seed oil content. The variation in its promoter and coding sequence leads to different natural alleles of this gene. The GmSWEET39 allelic effects on total oil content were confirmed in the seeds of soybean recombinant inbred lines, transgenic Arabidopsis, and transgenic soybean hairy roots. The frequencies of its superior alleles increased from wild soybean to cultivated soybean, and are much higher in released soybean cultivars. The findings herein suggest that the sequence variation in GmSWEET39 affects its relative expression and oil content in soybean seeds, and GmSWEET39 has been selected to increase seed oil content during soybean domestication and improvement.
Project description:Seeds of soybean (Glycine max L.) are a major source of plant-derived oils. In the past, improvements have been made in the quantity and quality of seed oil. Triacylglycerols (TAGs) are the principal components of soybean seed oil, and understanding the metabolic regulation of TAGs in soybean seeds is essential. Here, we identified four soybean genes encoding TAG lipases, designated as SUGAR DEPENDENT1-1 (GmSDP1-1), GmSDP1-2, GmSDP1-3 and GmSDP1-4; these are homologous to Arabidopsis thaliana SDP1 (AtSDP1). To characterize the function of these genes during grain filling, transgenic lines of soybean were generated via RNA interference to knockdown the expression of all four GmSDP1 genes. The seed oil content of the transgenic soybean lines was significantly increased compared with the wild type (WT). Additionally, fatty acid profiles of the WT and transgenic soybean lines were altered; the content of linoleic acid, a major fatty acid in soybean seeds, was significantly reduced, whereas that of oleic acid was increased in transgenic soybean seeds compared with the WT. Substrate specificity experiments showed that TAG lipase preferentially cleaved oleic acid than linoleic acid in the oil body membrane in WT soybean. This study demonstrates that the GmSDP1 proteins regulate both the TAG content and fatty acid composition of soybean seeds during grain filling. These results provide a novel strategy for improving both the quantity and quality of soybean seed oil.
Project description:Soybean is one of the most important oil crops in the world. Revealing the molecular basis and exploring key candidate genes for seed oil synthesis has great significance for soybean improvement. In this study, we found that oil accumulation rates and gene expression levels changed dynamically during soybean seed development. The expression levels of genes in metabolic pathways such as carbon fixation, photosynthesis, glycolysis, and fatty acid biosynthesis were significantly up-regulated during the rapid accumulation of oil in developing soybean seeds. Through weighted correlation network analysis, we identified six co-expression modules associated with soybean seed oil content and the pink module was the most positively correlated (r = 0.83, p = 7 × 10-4) network. Through the integration of differential expression and co-expression analysis, we predicted 124 candidate genes potentially affecting soybean seed oil content, including seven genes in lipid metabolism pathway, two genes involved in glycolysis, one gene in sucrose metabolism, and 12 genes belonged to transcription factors as well as other categories. Among these, three genes (GmABI3b, GmNFYA and GmFAD2-1B) have been shown to control oil and fatty acid content in soybean seeds, and other newly identified candidate genes would broaden our knowledge to understand the molecular basis for oil accumulation in soybean seeds.
Project description:Genes involved in fatty acids biosynthesis, modification and oil body formation are expected to be conserved in structure and function in different plant species. However, significant differences in the composition of fatty acids and total oil contents in seeds have been observed in different plant species. Comparative genomics was performed on 261 genes involved in fatty acids biosynthesis, TAG synthesis, and oil bodies formation in Arabidopsis, Brassica rapa, castor bean and soybean. In silico expression analysis revealed that stearoyl desaturase, FatB, FAD2, oleosin and DGAT are highly abundant in seeds, thereby considered as ideal candidates for mining of favorable alleles in natural population. Gene structure analysis for major genes, ACCase, FatA, FatB, FAD2, FAD3 and DGAT, which are known to play crucial role in oil synthesis revealed that there are uncommon variations (SNPs and INDELs) which lead to varying content and composition of fatty acids in seed oil. The predicted variations can provide good targets for seed oil QTL identification, understanding the molecular mechanism of seed oil accumulation, and genetic modification to enhance seed oil yield in plants.
Project description:Increasing seed oil content is one of the most important breeding goals for soybean due to a high global demand for edible vegetable oil. However, genetic improvement of seed oil content has been difficult in soybean because of the complexity of oil metabolism. Determining the major variants and molecular mechanisms conferring oil accumulation is critical for substantial oil enhancement in soybean and other oilseed crops. In this study, we evaluated the seed oil contents of 219 diverse soybean accessions across six different environments and dissected the underlying mechanism using a high-resolution genome-wide association study (GWAS). An environmentally stable quantitative trait locus (QTL), GqOil20, significantly associated with oil content was identified, accounting for 23.70% of the total phenotypic variance of seed oil across multiple environments. Haplotype and expression analyses indicate that an oleosin protein-encoding gene (GmOLEO1), colocated with a leading single nucleotide polymorphism (SNP) from the GWAS, was significantly correlated with seed oil content. GmOLEO1 is predominantly expressed during seed maturation, and GmOLEO1 is localized to accumulated oil bodies (OBs) in maturing seeds. Overexpression of GmOLEO1 significantly enriched smaller OBs and increased seed oil content by 10.6% compared with those of control seeds. A time-course transcriptomics analysis between transgenic and control soybeans indicated that GmOLEO1 positively enhanced oil accumulation by affecting triacylglycerol metabolism. Our results also showed that strong artificial selection had occurred in the promoter region of GmOLEO1, which resulted in its high expression in cultivated soybean relative to wild soybean, leading to increased seed oil accumulation. The GmOLEO1 locus may serve as a direct target for both genetic engineering and selection for soybean oil improvement.