Progress 06/01/03 to 05/31/08
Outputs
OUTPUTS: Presentations Bachlava E J.W. Burton, C. Brownie, S. Wang, J. Auclair and A. J. Cardinal. Quantitative trait loci mapping for oleic acid seed content in soybean. American Society of Agronomy Annual Meeting, November 4-8, 2007, New Orleans, LA. Bachlava E., R.E. Dewey, J.W. Burton, A.J. Cardinal. Mapping the microsomal omega-6 fatty acid desaturase genes controlling oleic acid seed content in soybean. The 98th America Oil Chemists' Society annual meeting & expo, May 13-16, 2007, Quebec City, Canada. Community resources generated Three F5-derived populations with phenotypic information and genotypic information will be released. Dr Nelson (USDA soybean curator) mentioned that they are not interested in keeping mapping populations in the collection. I will increase seed of these populations in the next couple of years and keep them in storage for the next 5 to 10 years to have seed available to interested public researchers. Poster Presentations Bachlava E., R.E. Dewey, J.W. Burton and A.J. Cardinal. Cosegregation of candidate genes for oleate biosynthesis with quantitative trait loci for oleic acid seed content in soybean. ASA-CSSA-SSSA annual meeting, October 5-9th, 2008, Houston, TX. Bachlava E. and A.J. Cardinal. Temperature and oleic acid seed content: Correlation in soybean populations with different maturity profiles. ASA-CSSA-SSSA annual meeting, October 5-9th, 2008, Houston, TX. Bachlava E., J. Auclair, J. Burton, R. E. Dewey, A. J. Cardinal. Genetic Control of High Oleic acid content in Soybean. Plant and Animal Genome XVI. The international Conference of the Status of Plant & Animal Genome Research. January 12-16, 2008, San Diego, CA. Abstracts Guide p. 217. Bachlava E., J. Auclair, J. Burton, A. J. Cardinal. Heritability of Oleic and Linolenic Acid Seed Content and Their Genetic Correlations with Quality and Agronomic Traits in Soybean. American Society of Agronomy International Annual Meeting, November 4-8, 2007, New Orleans, LA. http://a-c-s.confex.com/a-c-s/2007am/techprogram/S4012.HTM. Bachlava E., R.E. Dewey, J.W. Burton, A.J. Cardinal. Single Nucleotide Polymorphisms for the microsomal omega-6 desaturases in soybean. American Society of Agronomy Annual Meeting, November 12-16, 2006, Indianapolis, IN. Training Postdoctoral fellow. Jerome Auclair. Phenotypic and genotypic information on one population. PI performed the phenotypic analysis and QTL analysis since Auclair has left the program. PI will perform the final QTL analysis when SNP genotypic information is available. Eleni Bachlava (partly funded by this project) graduated with a Ph.D. in May, 2008. Phenotypic and genotypic information, and QTL analysis, candidate genes sequencing and development of allele specific primers, candidate gene mapping, and QTL mapping in two populations. Eleni Bachlava was awarded the AOCS Biotechnology Student Excellence Award (2nd): "Mapping the microsomal omega-6 fatty acid desaturase genes controlling oleic acid seed content in soybean" 98th AOCS annual meeting & Expo. Quebec City, QC, Canada (May 13-16th, 2007). Eleni has presented our work in numerous meetings and was the first author in all the 5 manuscripts derived from her work. PARTICIPANTS: PI (Cardinal): Developed the whole project, developed the field design with help from Dr. Brownie (NCSU statistician), analyzed phenotypic and genotypic data, and performed QTL mapping of one population. Tutored Jerome Auclair and Eleni Bachlava in the analysis of the data and several issues that arose during the project. Training: Postdoctoral fellow. Jerome Auclair. Phenotypic and genotypic information on one population. PI performed the phenotypic analysis and QTL analysis since Auclair has left the program. PI will perform the final QTL analysis when SNP genotypic information is available. Eleni Bachlava (partly funded by this project) graduated with a Ph.D. in May, 2008. Phenotypic and genotypic information, and QTL analysis, candidate genes sequencing and development of allele specific primers, candidate gene mapping, and QTL mapping in two populations. Eleni Bachlava was awarded the AOCS Biotechnology Student Excellence Award (2nd): "Mapping the microsomal omega-6 fatty acid desaturase genes controlling oleic acid seed content in soybean" 98th AOCS annual meeting & Expo. Quebec City, QC, Canada (May 13-16th, 2007). Eleni has presented our work in numerous meetings and was the first author in all the 5 manuscripts derived from her work. Eleni visited was trained to perform Tilling by a group in Collaborators: Dr. Dewey provided laboratory equipment and training of Eleni Bachlava in gene sequencing techniques and sequence data analysis. Dr. Joseph Burton provided laboratory equipment and training for fatty acid analysis. Dr. Perry Cregan laboratory will perform SNP genotyping of one population. TARGET AUDIENCES: Soybean breeders working with the oleic acid trait. Plant geneticists that work with the fatty acid pathway. PROJECT MODIFICATIONS: Delay in hiring post-doc fellow (9 months). Field problems (herbicide damage one year, and drought).
Impacts
We met all the objectives of the proposal. We have identified several QTLs and validated 1 associated with increased oleic acid content. In one population, we indentified QTLs on Linkage Groups (LG) N, A2, F, I, A1, and E. On a second realted population, we identified QTLs on LG M, G, D2, O, I, F. QTL on LG F was validated in both populations. We identified some environment sensitive QTLs. One epistatic interaction was detected between the QTL on LG F and the candidate gene FAD2-1B. We developed allele specific primers for the FAD2-1A, FAD2-1B, FAD2-2A, FAD2-2B candidate genes encoding the omega-6 microsomal desaturase and mapped them to linkage groups O, I, and L, respectively. We also developed SSR primers that are very close (1,870-42,587 bp) to candidate genes FAD2-2C, FAD2-2D, FAD6, AAPT1a, and AAPT1b that encode 2 constitutive omega-6 microsomal desaturases, the plastidial omega-6 microsomal desaturase, and 2 aminoalcoholphosphotransferases, respectively. The FAD2-2C, and FAD2-2D gene mapped to LG E and N, respectively. FAD6 and AAPT1a are linked and mapped to LG D1b. We could not map AAPT1b. We determined that QTLs co-segregate with FAD2-1B, AAPT1a, and FAD2-2C, indicating that differences in the parental alleles of these genes may cause changes in oleic acid content. However, these results need to be confirmed. All the other QTLs do not co-segregate with the other candidate genes. We have not published the results of our QTL mapping analysis in a second very large population (719 F4-derived lines) from this project. Multiple Interval analysis detected 4 QTLs on linkage groups A1, C1, D1b, and L. The combined QTLs explained a small proportion of the genetic variation (16.5%), indicating that many QTL had not been detected in this population because we only genotyped 90 SSR markers(funding restriction). We have requested that ~580 lines from this population are genotyped with SNPs (collaboration with Dr. Cregan) with funding from a United Soybean Board project in progress. We will have 300 to 400 SNPs markers for ~580 lines to perform a thorough QTL analysis. We discovered that the great majority of the genetic loci responsible for conferring a high oleic acid phenotype did not correspond to natural allelic variation of genes from the primary fatty acid biosynthetic pathway and that the role of the FAD2 genes in the genetic control of the high oleic acid phenotype inherited from N98-4445A is very small. Our findings contradict results from similar investigations in other oilseed crops which nearly always found clear associations between high oleic acid and specific metabolic genes involved in fatty acid biosynthesis. The continuation of the groundwork laid by this project will lead to new insights into the mechanisms by which plants (soybean) define and regulate the fatty acid composition of the seed oil.
Publications
- Meetings Abstracts Bachlava E., R.E. Dewey, J.W. Burton and A.J. Cardinal. Cosegregation of candidate genes for oleate biosynthesis with quantitative trait loci for oleic acid seed content in soybean. ASA-CSSA-SSSA annual meeting, October 5-9th, 2008, Houston, TX.
- Bachlava E. and A.J. Cardinal. Temperature and oleic acid seed content: Correlation in soybean populations with different maturity profiles. ASA-CSSA-SSSA annual meeting, October 5-9th, 2008, Houston, TX.
- Bachlava E., J. Auclair, J. Burton, R. E. Dewey, A. J. Cardinal. Genetic Control of High Oleic acid content in Soybean. Plant and Animal Genome XVI. The international Conference of the Status of Plant & Animal Genome Research. January 12-16, 2008, San Diego, CA. Abstracts Guide p. 217. Bachlava E., R.E. Dewey, J.W. Burton, A.J. Cardinal. Mapping the microsomal omega-6 fatty acid desaturase genes controlling oleic acid seed content in soybean. The 98th America Oil Chemists Society annual meeting & expo, May 13-16, 2007, Quebec City, Canada.
- Publications Bachlava E., R. E. Dewey, J. W. Burton, and A. J. Cardinal. 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., R. E. Dewey, J. W. Burton, A. J. Cardinal. 2009. Mapping candidate genes for oleate biosynthesis and their association with unsaturated fatty acid seed content in soybean. Mol. Breeding 23:337-347.
- Bachlava E., J. W. Burton, C. Brownie, S. Wang, J. Auclair, and A. J. Cardinal. 2008. Heritability of Oleic Acid Content in Soybean Seed Oil and Its Genetic Correlation with Fatty Acid and Agronomic Traits. Crop Sci. 48:1764-1772.
- Bachlava E., R. E. Dewey, J. Auclair, S. Wang, J. W. Burton, and A. J. Cardinal. 2008. Mapping Genes Encoding Microsomal ω-6 Desaturase Enzymes and Their Cosegregation with QTL Affecting Oleate Content in Soybean. Crop Sci. 48:640-650.
- Bachlava E., and Andrea J. Cardinal. 2009. Correlation between Temperature and Oleic Acid Seed Content in Three Segregating Soybean Populations. In Press: Crop Science.
- Bachlava E., J. Auclair, J. Burton, A. J. Cardinal. Heritability of Oleic and Linolenic Acid Seed Content and Their Genetic Correlations with Quality and Agronomic Traits in Soybean. American Society of Agronomy International Annual Meeting, November 4-8, 2007, New Orleans, LA. (Verified 1/1/2008, http://a-c-s.confex.com/a-c-s/2007am/techprogram/P32906.HTM).
- Bachlava E., J. Burton, R. E. Dewey, and A. J. Cardinal. Quantitative Trait Loci Mapping for Oleic Acid Seed Content in Soybean. American Society of Agronomy International Annual Meeting, November 4-8, 2007, New Orleans, LA. (Verified 1/1/2008, http://a-c-s.confex.com/a-c-s/2007am/techprogram/P32907.HTM).
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Progress 10/01/05 to 09/30/06
Outputs
Objectives 1) To develop allele-specific markers of candidate genes to investigate the relationship between quantitative trait loci (QTLs) for oleate content in the soybean oil and specific genes of the pathway and their interactions with the environment; 2)to detect single nucleotide polymorphisms (SNPs) and develop allele-specific markers for the isoforms of Fad2-1 gene; 3) to localize Fad2-1A and Fad2-1B in the soybean genome; and 4) to determine the association of the isoforms of Fad2-1 gene with the eate trait. Materials & Methods. Plant material, Phenotyping, and Genotyping. Population FAF00 consisting of 118 F5:7 lines derived from the cross of the high-oleate, low-linolenate line N97-3363-3, and the mid-oleate PI423893; and population FAE00 consisting of 724 F5:7 lines derived from the cross of the high-oleic, low-linolenic line N98-4445, and low palmitate, low linolenate, Satelite were planted in a sets in rep designs in Kinston, Clinton, and Plymotuh in 2005
and 2006. Fatty acid composition of seed oil was determined by gas-liquid chromatography of fatty acid methyl esters (2006 FAE00 samples remained to be done). 98 polymorphic SSR markers were genotyped in FAF00 from the 20 linkage groups of soybean genome. Genotyping in FAE00 is in progress. Oleate trait means were estimated by best linear unbiased prediction (BLUP) with SAS 9.1 and linkage analysis was performed with JoinMap 3.0 in population FAF00. Single Nucleotide Polymorphisms' detection. Sequence information was derived from GenBank (accessions AB188250 and AB188251) and isoform-specific primers were developed with Primer3. Two isoforms, Fad2-1A and Fad2-1B, were amplified, TA cloned into vectors and sequenced for all the parental lines and a control (Brim). ClustalW was used for sequence alignment and SNPs' detection. One of the SNPs detected for PI423893 in Fad2-1B was located on the restriction site of HpyCH4III endonuclease. A cleaved amplified polymorphic sequence (CAPS)
marker was designed and used for genotyping of FAF00 population. Fad2-1A was genotyped in FAF00 population with the allele-specific primer extension (ASPE) assay in a Luminex platform. Results Two SNP were detected for PI423893 in Fad2-1A coding region and upstream region, respectively. The SNP in Fad2-1A coding region changes the amino acid residue but no significant association was found between Fad2-1A and the oleate trait in FAF00 (preliminary analysis). Fad2-1A is linked to SSR markers satt153 and sat_108 on LG O. Five SNPs were detected for PI423893 in Fad2-1B coding region and only two of those SNPs result in a non-synonymous amino acid change. No significant association was found between the Fad2-1B CAPS marker and oleate trait in FAF00 (preliminary analysis). Fad2-1B maps to LG I within SSR markers satt354 and sat_268. Discussion Neither Fad2-1A nor Fad2-1B mapped close to QTL for oleate trait reported in SoyBase. The lack of association between these loci and oleate in FAF00
implies that these isoforms cannot explain the observed variation in oleic acid content. The location of the SNP in Fad2-1A coincides with a region responsible for the enzyme's instability at high temperatures.
Impacts
Cosegregation analysis between QTLs for high oleic acid and candidate genes will be used to test the importance of structural genes in the regulation of oleic acid synthesis in soybeans. If a candidate gene segregates independently of QTLs, this would indicate that other independent genes, such as trans-acting regulatory genes or transcription factors, are important for oleic acid synthesis. The study of QTL-by-environment interactions involving particular QTLs and candidate genes will help us understand which steps of the biosynthetic pathway are more sensitive to environmental changes. Such knowledge will be invaluable for plant breeders to assist manipulation of oleic acid composition. For example, if particular QTLs or candidate genes interact, maximum improvement for oleic acid content can be achieved only if particular allelic combinations are selected. Similarly, if particular QTLs or candidate genes are highly sensitive to changes in the environments, a breeder
may choose not to incorporate those loci in their selection criteria.
Publications
- Abstract. Bachlava E., R.E. Dewey, J.W. Burton, A.J. Cardinal. Single Nucleotide Polymorphisms for the Microsomal Omega-6 Desaturases in Soybean. CSSA-ASA Annual Meeting, 12-16 November, 2006, Indianapolis, IN. Poster Presentation. Bachlava E., R.E. Dewey, J.W. Burton, A.J. Cardinal. Single Nucleotide Polymorphisms for the Microsomal Omega-6 Desaturases in Soybean. ASA Annual Meeting, 12-16 November, 2006, Indianapolis, IN.
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