Progress 10/01/08 to 09/30/13
Outputs
Target Audience: Soybean reseachers and plant breeders interested in modified seed oil traits. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Graduate student training. How have the results been disseminated to communities of interest? Publication in peer-reviewed journals. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have previously shown that soybean germplasm lines containing decreased palmitic acid (16:0) content are associated with debilitating mutations in 16:0-ACP thioesterase genes. Specifically, we demonstrated that the low 16:0 loci referred to as fap3 and fapnc could be attributed to mutations in the predominant 16:0-ACP thioesterase gene, FATB1a. However, an additional low 16:0-conferring locus, fap1, is not associated with perturbations in any member of the small gene family encoding 16:0-ACP thioesterase activity. The mapping of molecular markers to populations segregating at fap1 led to the discovery of a 3-keto-acyl-ACP synthase III (KAS III) gene that resided in the vicinity of co-segregating markers. DNA sequence analysis of this KAS III locus by colleagues associated with the USDA-ARS suggested that a splice junction mutation occurs in individuals possessing the fap1 locus. We conducted Northern blot analyses on mutant versus normal 16:0-containing soybean lines. Our results suggested that unlike most splice-site mutations, the mutation found in this particular KAS III gene does not lead to an unstable transcript. On the contrary, a stable transcript is produced in which the first intron is retained and the remaining introns are faithfully removed. The mature transcript found in fap1-containing lines would thus be predicted to give rise to a nonfunctional protein product. The predictions we made based on RNA blotting were further confirmed by sequence analysis of the RT-PCR products derived from mutant versus normal soybean lines. This information should lead to the development of perfect molecular markers that could be used assist in the breeding of the low 16:0 trait. Our studies have also demonstrated that the two soybean seed-specific 18:1 desaturase enzymes encoded by the GmFAD2-1A and GmFAD2-1B genes are responsible for the great majority of polyunsaturated fatty acid production that occurs in soybean seeds. Because mutations in these genes represent the most direct way to obtain stable, elevated oleic acid phenotypes in the soybean oil, we proposed to screen Plant Introductions (PIs) in the US germplasm collection that have an oleic acid content greater than 33% (and are also from maturity groups 3 – 7) for potential mutations in these genes. We continued our efforts to understand the effect that a SNP in the FAD2-1b gene observed in several PIs in the soybean germplasm collection has on 18:1 content. These lines had a polymorphism in the coding region of the FAD2-1b gene that would be predicted to slightly diminish enzyme activity. PI423893 has this SNP in FAD2-1b and our published studies mapped a QTL for oleic acid with a small effect near this gene. We developed 2 Heterogeneous Inbred Families (HIF) from a 2 F5 plants heterozygous for the FAD2-1b gene with this SNP. The F5 plants were selected from F3:5 lines from the cross of N98-4445A × PI423893 (FAS population, Bachlava et al, 2008 and 2009). Seed from each heterozygous plants was planted in a 10 foot row and individual plants were tagged, genotyped for the FAD2-1b SNP, and harvested individually. One HIF family had 20 plants and the second HIF family had 19 plants. Fatty acid analysis from 5 seeds from each plant of the 2 HIFs was performed. Oleic acid content was significantly greater (3.4%) in the plants that had the FAD2-1b gene with the SNP from PI423893, confirming our previous QTL results. We have identified nucleotide polymorphisms in soybean KASII genes that are found in elevated palmitate germplasm developed by Walt Fehr's group. Specifically, two separate point mutations were found in the GmKAS IIB genes in the A25 and A30 germplasms, and point mutations in the GmKAS IIA gene were observed in a lines designated A21 and A27. To determine whether the observed mutations strictly correlate with the elevated 16:0 phenotypes, Dr. Fehr provided us with seed from F2 families of crosses between each of these lines and the cultivar Archer. Genomic DNAs were isolated from approximately twenty elevated 16:0 individuals and twenty phenotypically wild type individuals derived from each of these crosses. Genotyping these individuals for the candidate GmKAS II mutations established that these mutations co-segregated with the elevated 16:0 trait.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Keogh, M.R., Courtney, P.D., Kinney, A.J. and Dewey, R.E. (2009) Functional characterization of phospholipid N-methyltransferases from Arabidopsis and soybean. J. Biol. Chem. 284: 15439-15447.
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Bachlava, E., Dewey, R.E., Burton, J.W. and Cardinal, A.J. (2009) Mapping candidate genes for oleate biosynthesis and their association with the unsaturated fatty acid seed content in soybean. Mol. Breeding 23: 337-347.
- Type:
Journal Articles
Status:
Published
Year Published:
2009
Citation:
Bachlava, E., Dewey, R.E., Burton, J.W. and Cardinal, A.J. (2009) Mapping and comparison of quantitative trait loci for oleic acid seed content in two segregating soybean populations. Crop Sci. 49: 1-10.
- Type:
Book Chapters
Status:
Published
Year Published:
2010
Citation:
Dewey, R.E. and Zhang, P. (2010) Candidate gene analysis of mutant soybean germplasm. In K. D. Bilyeu, M.B. Ratnaparkhe, C. Kole, eds., Genetics, Genomics and Breeding of Soybean. CRC Press, Boca Raton, FL. pp. 187-197.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
De Vries, B.D., Fehr, W.R., Welke, G.A. and Dewey, R.E. (2011) Molecular analysis of the mutant alleles for elevated palmitate concentration in soybean. Crop Sci. 51: 2554-2560.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
Cardinal, A.J., Burton, J.W., Camacho-Roger, A.M., Whetten R., Chappell, A.S., Bilyeu, K.D., Auclair, J. and Dewey, R.E. (2011) Molecular analysis of GmFAD3A in two soybean populations segregating for the fan, fap1, and fapnc loci. Crop Sci. 51: 2104-2112.
- Type:
Journal Articles
Status:
Published
Year Published:
2011
Citation:
De Vries, B.D., Fehr, W.R., Welke, G.A. and Dewey, R.E. (2011) Molecular characterization of the mutant fap3 (A22) allele for reduced palmitate concentration in soybean. Crop Sci. 51: 1611-1616.
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Cardinal, A.J., Whetten, R., Wang, S., Auclair, J., Hyten, D., Cregan, P., Bachlava, E., Gillman, J., Rameriz, M., Dewey, R., Upchurch, G., Miranda, L. and Burton, J.W. (2013) Mapping the low palmitate fap1 mutation and validation of its effects in soybean oil and agronomic traits in three soybean populations. Theor. Appl. Genet. (Published on-line Oct. 17, 2013).
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Progress 10/01/11 to 09/30/12
Outputs
OUTPUTS: We have previously shown that soybean germplasm lines containing decreased palmitic acid (16:0) content are associated with debilitating mutations in 16:0-ACP thioesterase genes. Specifically, we demonstrated that the low 16:0 loci referred to as fap3 and fapnc could be attributed to mutations in the predominant 16:0-ACP thioesterase gene, FATB1a. However, an additional low 16:0-conferring locus, fap1, is not associated with perturbations in any member of the small gene family encoding 16:0-ACP thioesterase activity. The mapping of molecular markers to populations segregating at fap1 led to the discovery of a 3-keto-acyl-ACP synthase III (KAS III) gene that resided in the vicinity of co-segregating markers. DNA sequence analysis of this KAS III locus by colleagues associated with the USDA-ARS suggested that a splice junction mutation occurs in individuals possessing the fap1 locus. To investigate the molecular consequences of the splice-site mutation, we conducted Northern blot analyses on mutant versus normal 16:0-containing soybean lines. Our results suggested that unlike most splice-site mutations, the mutation found in this particular KAS III gene does not lead to an unstable transcript. On the contrary, a stable transcript is produced in which the first intron is retained and the remaining introns are faithfully removed. The mature transcript found in fap1-containing lines would thus be predicted to give rise to a nonfunctional protein product. The predictions we made based on RNA blotting were further confirmed by sequence analysis of the RT-PCR products derived from mutant versus normal soybean lines. This information should lead to the development of perfect molecular markers that could be used assist in the breeding of the low 16:0 trait. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Soybean in the predominant oilseed crop that is grown in the US. Much of the value of soybean is derived from its oil component, which is used in a multitude of food, feed and industrial applications. For many edible applications, the fatty acid composition of most commercially grown cultivars is not optimal. An increase in the oleic acid content of the seed that is accompanied by a concomitant decrease in the polyunsaturated linoleic and linolenic fatty acids would give rise to an oil with increased stability and shelf life. In addition, enhancement of the palmitic acid or stearic acid contents, saturated fatty acids, can increase the shelf life and stability even further. A high oleic acid, high saturate soybean oil would require less hydrogenation, a process that introduces undesirable trans fatty acids into the oil. Increasing the monounsaturated and saturated fatty acid contents could lead to soybean varieties with an improved oil profiles for a variety of feed and nonfeed applications, and thus improve the competitiveness of soybean oil in the global marketplace.
Publications
- No publications reported this period
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Progress 10/01/10 to 09/30/11
Outputs
OUTPUTS: We have identified nucleotide polymorphisms in soybean KASII genes that are found in elevated palmitate germplasm developed by Walt Fehr's group. Specifically, two separate point mutations were found in the GmKAS IIB genes in the A25 and A30 germplasms, and point mutations in the GmKAS IIA gene were observed in a lines designated A21 and A27. To determine whether the observed mutations strictly correlate with the elevated 16:0 phenotypes, Dr. Fehr provided us with seed from F2 families of crosses between each of these lines and the cultivar Archer. Genomic DNAs were isolated from approximately twenty elevated 16:0 individuals and twenty phenotypically wild type individuals derived from each of these crosses. Genotyping these individuals for the candidate GmKAS II mutations established that these mutations co-segregated with the elevated 16:0 trait. In a previous research project, we were successful in identifying the SACPD-C gene as being a major determinant in defining the 18:0 content of the soybean seed as evidenced by the fact that two independent elevated stearate germplasm sources (A6; 30% 18:0 and FAM94-41; 9 - 15% 18:0) possessed debilitating mutations in this gene. To achieve additional gains in 18:0 content, we thought it would be useful to identify additional elevated 18:0 sources where the gene(s) responsible for the trait are not allelic to SACPD-C. We analyzed two lines provided by Joe Burton: PI567719 (9.0% 18:0) and JWB03-806-7-19 (7.45%). The latter source was one recovered by Dr. Burton by screening an X-ray mutagenized soybean population. To test whether the elevated 18:0 phenotype of either of these lines is associated with mutations in SACPD-C, we conducted a DNA sequence analysis using PCR-amplified genomic DNAs from each germplasm. This analysis identified a novel, nonconservative mutation in the SACPD-C gene from PI567719 (i.e. a mutation different from that found in A6 or FAM94-41). In contrast, no mutations were observed in the SACPD-C gene from JWB03-806-7-19. The results from this analysis suggest that the causative mutation responsible for the elevated stearate phenotype from PI567719 is allelic to the mutations found in A6 and FAM94-41. However, it is likely that the mutation responsible for this phenotype in JWB03-806-7-19 occurs in a separate gene and therefore may have the potential of being combined (particularly with FAM94-41) toward development of agronomically useful elevated 18:0 soybean lines. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Soybean in the predominant oilseed crop that is grown in the US. Much of the value of soybean is derived from its oil component, which is used in a multitude of food, feed and industrial applications. For many edible applications, the fatty acid composition of most commercially grown cultivars is not optimal. An increase in the oleic acid content of the seed that is accompanied by a concomitant decrease in the polyunsaturated linoleic and linolenic fatty acids would give rise to an oil with increased stability and shelf life. In addition, enhancement of the palmitic acid or stearic acid contents, saturated fatty acids, can increase the shelf life and stability even further. A high oleic acid, high saturate soybean oil would require less hydrogenation, a process that introduces undesirable trans fatty acids into the oil. Increasing the monounsaturated and saturated fatty acid contents could lead to soybean varieties with an improved oil profiles for a variety of feed and nonfeed applications, and thus improve the competitiveness of soybean oil in the global marketplace.
Publications
- De Vries, B.D., Fehr, W.R., Welke, G.A. and Dewey, R.E. (2011) Molecular characterization of the mutant fap3 (A22) allele for reduced palmitate concentration in soybean. Crop Sci. 51: 1611-1616.
- Cardinal, A.J., Burton, J.W., Camacho-Roger, A.M., Whetten R., Chappell, A.S., Bilyeu, K.D., Auclair, J. and Dewey, R.E. (2011) Molecular analysis of GmFAD3A in two soybean populations segregating for the fan, fap1, and fapnc loci. Crop Sci. 51: 2104-2112.
- De Vries, B.D., Fehr, W.R., Welke, G.A. and Dewey, R.E. (2011) Molecular analysis of the mutant alleles for elevated palmitate concentration in soybean. Crop Sci. 51: 2554-2560.
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Progress 10/01/09 to 09/30/10
Outputs
OUTPUTS: During this past year we have continued our efforts to survey PIs in the soybean germplasm collection that display elevated 18:1 phenotypes for mutation in FAD2-1 genes. We grew 24 PIs and 7 checks for a 3rd year experiment in Clayton at two planting dates (middle of May and middle of June). There were 4 PIs that matured as early MG3 lines and had an oleate content between 37 to 48% in the first planting date and 31 to 38% in the second planting date averaged across three years. The oleate content in these lines decreased in the second planting date by more than 3% up to 17% points. This group includes PI 603452 that has a mutation in FAD2-1a that leads to a frame shift and premature truncation of the protein. In addition, results from quantitative PCR of the steady-state transcript abundance for the FAD2-1 genes showed that FAD2-1a mRNA levels in PI 603452 were reduced 10-fold when compared to a normal line (Dr. Upchurch, unpublished data). These results support the conclusion that the mutation in the FAD2-1a gene causes the increased oleate content phenotype observed in PI 603452. There were 14 PIs that matured as late MG4 to early MG5 lines and had an oleate content between 29 to 42% in the first planting date and 26 to 37% in the second planting averaged across 3 years. There were a few lines in which the oleate content did not decrease by more than 1% in the second planting date and 3 lines that the oleate content increased in the second planting date. These lines had polymorphisms in the coding regions of FAD2-1a and FAD2-1b genes that would be predicted to have no effect on enzyme activity or that may slightly diminish enzyme activity. There were 5 PIs that matured as late MG5 lines and had an oleate content between 29 to 39% in the first planting date and 32 to 39% in the second planting date. These lines had polymorphisms in the coding regions of FAD2-1a and FAD2-1b genes that would be predicted to have no effect on enzyme activity or that may slightly diminish enzyme activity. We also continued our efforts to understand the effect that a SNP in the FAD2-1b gene observed in several PIs in the soybean germplasm collection has on 18:1 content. These lines had a polymorphism in the coding region of the FAD2-1b gene that would be predicted to slightly diminish enzyme activity. PI423893 has this SNP in FAD2-1b and our published studies mapped a QTL for oleic acid with a small effect near this gene. We developed 2 Heterogeneous Inbred Families (HIF) from a 2 F5 plants heterozygous for the FAD2-1b gene with this SNP. The F5 plants were selected from F3:5 lines from the cross of N98-4445A and PI423893. Seed from each heterozygous plants was planted in a 10 foot row and individual plants were tagged, genotyped for the FAD2-1b SNP, and harvested individually. One HIF family had 20 plants and the second HIF family had 19 plants. Fatty acid analysis from 5 seeds from each plant of the 2 HIFs was performed. Oleic acid content was significantly greater (3.4%) in the plants that had the FAD2-1b gene with the SNP from PI423893, confirming our previous QTL results. PARTICIPANTS: Nothing significant to report during this reporting period. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Soybean in the predominant oilseed crop that is grown in the US. In 2208, soybean was grown on over 75 million acres in the US, with a total yield of nearly 3 billion bushels. Much of the value of soybean is derived from its oil component, which is used in a multitude of food, feed and industrial applications. For many edible applications, the fatty acid composition of most commercially grown cultivars is not optimal. An increase in the oleic acid content of the seed that is accompanied by a concomitant decrease in the polyunsaturated linoleic and linolenic fatty acids would give rise to an oil with increased stability and shelf life. Furthermore, high oleic acid soybean oil would require less hydrogenation, a process that introduces undesirable trans fatty acids into the oil. The development of high oleic acid soybean cultivars, as outlined in this project, could lead to soybean varieties with an improved oil profile that would make food products derived from it more healthy, better suited for the production of biofuels, and thus improve the competitiveness of soybean oil in the global marketplace.
Publications
- Dewey, R.E. and Zhang, P. (2010) Candidate gene analysis of mutant soybean germplasm. In K. D. Bilyeu, M.B. Ratnaparkhe, C. Kole, eds., Genetics, Genomics and Breeding of Soybean. CRC Press, Boca Raton, FL. pp. 187-197.
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Progress 10/01/08 to 09/30/09
Outputs
OUTPUTS: Our previous studies have demonstrated that the two soybean seed-specific 18:1 desaturase enzymes encoded by the GmFAD2-1A and GmFAD2-1B genes are responsible for the great majority of polyunsaturated fatty acid production that occurs in soybean seeds. Because mutations in these genes represent the most direct way to obtain stable, elevated oleic acid phenotypes in the soybean oil, we proposed to use TILLING technologies to identify new mutations in these genes. We also identified 132 Plant Introductions (PIs) in the US germplasm collection that have an oleic acid content greater than 33% (and are also from maturity groups 3 - 7). During this past year we conducted both fatty acid and molecular analyses on ~90 of these PIs to determine which of this lines may have increased oleate phenotypes that are attributable to mutations in either GmFAD2-1A and GmFAD2-1B. In general, relatively little sequence polymorphism was observed within the FAD2-1A gene. A T to C substitution at position 990 (with respect to the start codon of the cDNA) was observed in some of the PIs, but because this substitution is "silent" and does not alter an amino acid, it is unlikely to have any negative impact on gene function. We also found several lines that possess a G to C polymorphism at position 64 that changes the Gly amino acid at position 22 to an Arg residue. However, this polymorphism does not appear to negatively affect gene function. The most promising FAD2-1A mutation was a frame shift mutant in PI603452 that completely eliminates gene function. This mutant line was previously discovered by Kristen Bilyeu, USDA-ARS, Columbia, Missouri. In contrast, polymorphisms at a total of 11 positions were observed in the FAD2-1B sequence. The most promising mutants are the following: Germplasm lines PI210179 and PI283327 possess a C to G substitution at position 409 that changes Pro 137 to an Arg amino acid. PI567189 and PI578451 contain T to C substitutions at position 143 that changes Ile 143 to a Thr residue. Both of these polymorphisms are nonconservative and would be likely to negatively impact enzyme/gene function. Recent communications with Kristen Bilyeu suggest that she has discovered these mutations as well, and also has evidence that they are detrimental to function. Another interesting class of polymorphisms was found in accessions PI612614 and PI408246-1. The FAD2-1B gene in both of these PIs possess two unique polymorphisms, a C to T substitution at position 636 that is silent, and a T to A polymorphism that changes the Leu amino acid at position 242 to a Met residue. This substitution is conservative, however, and we do not believe that it impacts gene function. In addition to the above-mentioned PIs, we conducted a similar FAD2-1 analysis from 6 TILLING lines were received from Khalid Meksem that had been screened by the Meksem lab for mutations in the FAD2-1A or FAD2-1B genes. In five of the six lines, we could find no mutations in FAD2-1 genes. A true EMS-induced mutation was observed in the FAD2-1B gene of the remaining line, but this mutation was silent, involving the modification of the G residue at position 753 to an A nucleotide. PARTICIPANTS: Not relevant to this project. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Soybean in the predominant oilseed crop that is grown in the US. In 2208, soybean was grown on over 75 million acres in the US, with a total yield of nearly 3 billion bushels. Much of the value of soybean is derived from its oil component, which is used in a multitude of food, feed and industrial applications. For many edible applications, the fatty acid composition of most commercially grown cultivars is not optimal. An increase in the oleic acid content of the seed that is accompanied by a concomitant decrease in the polyunsaturated linoleic and linolenic fatty acids would give rise to an oil with increased stability and shelf life. Furthermore, high oleic acid soybean oil would require less hydrogenation, a process that introduces undesirable trans fatty acids into the oil. The development of high oleic acid soybean cultivars, as outlined in this project, could lead to soybean varieties with an improved oil profile that would make food products derived from it more healthy, better suited for the production of biofuels, and thus improve the competitiveness of soybean oil in the global marketplace.
Publications
- Bachlava, E., Dewey, R.E., Burton, J.W. and Cardinal, A.J. (2009) Mapping and comparison of quantitative trait loci for oleic acid seed content in two segregating soybean populations. Crop Sci. 49: 1-10.
- Bachlava, E., Dewey, R.E., Burton, J.W. and Cardinal, A.J. (2009) Mapping candidate genes for oleate biosynthesis and their association with the unsaturated fatty acid seed content in soybean. Mol. Breeding 23: 337-347.
- Keogh, M.R., Courtney, P.D., Kinney, A.J. and Dewey, R.E. (2009) Functional characterization of phospholipid N-methyltransferases from Arabidopsis and soybean. J. Biol. Chem. 284: 15439-15447.
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