Progress 05/28/13 to 05/08/18
Outputs
Progress Report Objectives (from AD-416): 1) Identify genetic loci which contribute positive alleles to seed yield and quality traits and which control variation in seed quality, especially seed oil, protein, carbohydrate content, and fatty acid composition of legume seeds; develop strategies to facilitate effective transfer of useful genes into cultivated legumes; 1a) Mapping and characterizing mutant alleles that contribute to variation in seed fatty acid composition; 1b) Discovery of the molecular identity of genes that influence overall protein or oil content by forward genetics approaches. 2) Determine epigenetic patterns in soybean seeds during development, assess relationships between chromatin state and gene expression, and develop approaches to modify gene expression in developing seeds; 2a) Genome-wide profiling of epigenetic state and gene expression in developing soybean seeds by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequence-based expression profiling (RNA- seq). 3) Evaluate the outcrossing and diversity of Phytophthora sojae populations and host plant interactions. 4) Evaluate the population dynamics of Fusarium virguliforme. 5) Organize, manage, and conduct the Northern Uniform Soybean Tests. Approach (from AD-416): The project uses molecular biological approaches to study the regulation of genes that are required for soybean seed development. Biochemical approaches are used to study the interactions and function of gene products that are important for the accumulation of storage proteins or fatty acids in seeds. Molecular and genetic tools are used to define genes and genetic intervals that contribute to seed protein and oil content in mutant plants. This is a five-year summary for the composition project that terminated in May 2018. Objective 1. A. Identification of genetic loci contributing to soybean fatty acid composition and B. Discovery of the molecular identity of genes that influence overall protein or oil composition. The project employed a forward genetics approach to screen for new genes and new alleles that influence or improve soybean fatty acid composition or the protein/oil content of soybean seeds in a mutant population developed by the research team. When the project initiated, the focus was on a group of 24 newly identified mutants with an unusual oil composition. Sixteen of these lines had elevated levels of oleic acid. It was determined that nine of these mutant lines with elevated oleic acid, plus an additional line that was subsequently identified, carried mutant alleles of the FAD2- 1A desaturase. It was also determined that one line from the initial 16 carried a mutation in the FAD2-1B gene, and that when combined the fad2- 1a fad2-1b double mutant could achieve up to 80% oleic acid in the seed oil fraction. The remaining six lines are still under investigation, and are still under study with the goal of identifying the mutant genes. In the course of the screen, six new alleles of the SACPD-C gene resulting in elevated stearic acid, 3 new alleles of the FAD3A gene, and one allele of the FAD3C gene resulting in low linolenic acid, and two new alleles of the FATB-1A gene resulting in low levels of palmitic acid were identified. Molecular markers for all of these mutations were developed, and numerous gene combinations that produce improved oil profiles (for example high oleic-low linolenic) were evaluated. These lines provide breeders with alternative conventional germplasm. A patent application was submitted for one combination for high oleic soybeans. Several novel loci (high linolenic acid, high stearic acid that is not due to SACPD-C mutation) are also still under investigation. These lines are a valuable resource for the continued improvement of soybean oil composition. Research focused on aspects of soybean fatty acid composition, as well as the selection and evaluation of protein and oil composition mutants. This trait is more qualitative and requires an extra generation to measure composition of bulk seed. Scientists followed a number of promising lines in the field for multiple seasons, performed mapping crosses and backcrosses and evaluated progeny, performed preliminary mapping, and evaluated several methodologies for mapping and mutation discovery in soybean. The molecular identity of the loci influencing protein and oil content and the mechanism of nutrient partitioning in developing seed are the subject of investigation for a future project. The mutant population was further leveraged as a resource and made available for screening by collaborators for various other traits, such as seed ionomic composition, time to flowering and maturity, and soybean architectural traits. Reverse genetic approaches were employed to improve soybean meal by screening the population for mutations that reduce levels of undigestible carbohydrates. Objective 2. Determine epigenetic patterns, modify gene expression in soybean cotyledons. In two related experiments, the network of gene regulation controlling soybean leaf and cotyledon senescence was explored. In the first approach, patterns of gene expression during senescence between soybean leaves and cotyledons were compared. A set of soybean senescence genes was defined and the identity of important transcriptional regulators was inferred based on observed patterns and known DNA binding sites. The study confirmed the importance of several transcription factor families in both cotyledon and leaf senescence, and highlighted differences in the mobilization of nutrient reserves between the two tissues. This work was extended to study how the natural transcriptional program of senescence is deferred in soybean leaves from �sink-limited� soybean plants, plants that have had developing pods artificially removed, or that fail to make seeds due to flower sterility. Evidence suggests that the role of lipoxygenase activity in sink-limited tissues was not strictly limited to plant defense, but may be involved in nutrient storage and metabolism. A new member of the soybean �stay-green� gene family that may be involved in the regulation of chlorophyll degradation was identified. These results increase understanding of normal senescence, early senescence in response to drought or pathogen attack, and to the phenomenon of �stay-green� soybeans. Some of these genes and transcriptional relationships warrant further investigation in future work which will likely be pursued in collaboration with other researchers. Objective 3. Evaluate the outcrossing and diversity of Phytophthora sojae populations and host plant interactions. A total of ~600 Phytophthora sojae isolates, the pathogen that causes soybean root and stem rot, from Indiana and other states have been assembled by ARS scientist in West Lafayette, Indiana. Over half of the isolates have been pathotyped using differentials carrying Rps genes. New differentials carrying recently discovered Rps genes, Rps11, RpsUN1 and RpsUN2 were obtained. These new differentials were seed-increased in greenhouse and are being incorporated into P. sojae pathotyping. In addition to P. sojae, another pathogen, P. sansomeana, also causes Phytophthora root rot of soybean. ARS scientist in West Lafayette, Indiana, sequenced the genome of the type strain of this pathogen, 1819B, and a draft genome assembly is available. The complete mitochondrial genome of this pathogen was also assembled. Objective 4. Evaluate the population dynamics of Fusarium virguliforme. Data mining was used to build an informative microsatellite database for Fusarium virguliforme, the fungal pathogen that causes sudden death syndrome of soybean. Experiments were conducted to validate these new microsatellite markers. These markers are now being used to examine global population structure of F. virguliforme. Additionally, the genome of the type strain of F. virguliforme, Mont-1, was sequenced and the complete genome of its mitochondrion was assembled. Objective 5. Organize, manage, and conduct the Northern Uniform Soybean Tests. The 2017 Northern Uniform Soybean Tests were successfully completed and the 2018 tests are in progress. In the 2017 tests, 407 soybean breeding lines and checks in maturity groups 00 to IV were evaluated and the tests were conducted at 49 sites in 10 Midwest states in the United States and two provinces in Canada. These soybean lines were evaluated for yield and other agronomic characteristics including seed quality, lodging, and shattering, etc. They were also evaluated for disease resistance including soybean cyst nematode, Phytophthora root and stem rot, green stem, and iron chlorosis, etc. Soybean lines bred for seed quality traits (oil and protein content, amino acid, high oleic acid) were evaluated for their target trait. These breeding lines were submitted by public breeders in the Unites States and Canada. ARS scientist in West Lafayette, Indiana, directly conducted tests at three sites in Indiana and evaluated all 407 lines for resistance to Phytophthora root rot under greenhouse conditions. Data were collected from all participants and analyzed. The results were published in the book �THE UNIFORM SOYBEAN TESTS NORTHERN REGION 2017.� The hard copy was delivered to participants and interested stakeholders. The electronic copy of the book is freely available online. Accomplishments 01 Publication of the book �The Uniform Soybean Tests Northern Region 2017. " Public soybean breeders lack the resource to evaluate their breeding lines in multiple locations and in diverse environments. The ARS lab in West Lafayette, Indiana, organizes the Northern Uniform Soybean Tests yearly. These tests provide the information required by public breeders to accurately assess a line�s ability to produce prior to its potential release, while eliminating the constraints of individually conducting trials in numerous locations and across multiple states. The Northern Uniform Soybean Tests evaluate soybean breeding lines for agronomic performance, disease resistance and seed quality traits in Midwest states in the United States and provinces in Canada. The soybean lines are those developed by public breeders in the United States and Canada that are suitable for planting in these regions. In 2017, 407 soybean breeding lines and checks in maturity groups 00 to IV were evaluated. Eighteen university and government scientists participated and the tests were conducted at 49 sites in 10 Midwest states in the United States and two provinces in Canada. In addition to performing tests at multiple sites in Indiana, the ARS lab in West Lafayette, Indiana, collected data from participants, analyzed the data, and published the 2017 test results in this book. The hard copy was delivered to participants and interested stakeholders. The electronic copy of the book is freely available online. This book is used as primary evidence by breeders when making the decision to further advance their lines or release their lines to the public.
Impacts
(N/A)
Publications
- Wickland, D.P., Battu, G., Hudson, K.A., Diers, B.W., Hudson, M.E. 2017. A comparison of genotyping-by-sequencing analysis methods on low-coverage crop datasets shows advantages of a new workflow, GB-eaSy. BMC Bioinformatics. 18:586.
- Zhang, N., Cai, G., Price, D.C., Crouch, J., Gladieux, P., Hillman, B., Khang, C.H., Lebrun, M., Lee, Y., Luo, J., Qiu, H., Veltri, D., Wisecarver, J.H., Zhu, J., Bhattacharya, D. 2018. Genome wide analysis of the transition to pathogenic lifestyles in Magnaporthales fungi. Scientific Reports. 8:5862.
- Thapa, R., Carrero-Colon, M., Addo-Quaye, C., Held, J., Dilkes, B., Hudson, K.A. 2018. New alleles of FAD3A confer reduced linolenic acid trait to soybean seeds. Crop Science. 58(2):713-718.
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Progress 10/01/16 to 09/30/17
Outputs
Progress Report Objectives (from AD-416): 1) Identify genetic loci which contribute positive alleles to seed yield and quality traits and which control variation in seed quality, especially seed oil, protein, carbohydrate content, and fatty acid composition of legume seeds; develop strategies to facilitate effective transfer of useful genes into cultivated legumes; 1a) Mapping and characterizing mutant alleles that contribute to variation in seed fatty acid composition; 1b) Discovery of the molecular identity of genes that influence overall protein or oil content by forward genetics approaches. 2) Determine epigenetic patterns in soybean seeds during development, assess relationships between chromatin state and gene expression, and develop approaches to modify gene expression in developing seeds; 2a) Genome-wide profiling of epigenetic state and gene expression in developing soybean seeds by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequence-based expression profiling (RNA- seq). 3) Evaluate the outcrossing and diversity of Phytophthora sojae populations and host plant interactions. 4) Evaluate the population dynamics of Fusarium virguliforme. 5) Organize, manage, and conduct the Northern Uniform Soybean Tests. Approach (from AD-416): The project uses molecular biological approaches to study the regulation of genes that are required for soybean seed development. Biochemical approaches are used to study the interactions and function of gene products that are important for the accumulation of storage proteins or fatty acids in seeds. Molecular and genetic tools are used to define genes and genetic intervals that contribute to seed protein and oil content in mutant plants. Objective 1a. Identification of genetic loci contributing to soybean fatty acid composition. We continue to advance several double and higher order mutant combinations that carry defects in multiple genes in the fatty acid biosynthetic pathway. We are working to create combinations including our high oleic mutations with additional gene mutations to obtain and evaluate a convention high oleic low-linolenic line. We have also made significant progress in combining mutations which confer a high stearic acid trait and the low linolenic trait. A soybean oil high in stearic acid would provide an alternative to the partial hydrogenation or esterification of soybean oil to obtain solid fats for a variety of food products. We have manuscripts in preparation to describe these combinations and the new alleles to reduce linolenic acid. We are still mapping several unidentified loci that affect oleic acid levels, linolenic acid levels, and palmitic acid levels. We have mapped a morphological mutant to a 3Mb interval on chromosome 17 and have identified a candidate gene. In a related subordinate project funded by the United Soybean Board, we have made crosses to integrate a new allele of raffinose synthase3 into our high oleic and low linolenic background. We need another season to determine the effect of this mutation on seed carbohydrate composition. We are likely to submit a manuscript describing this allele, and our reverse genetics methods in FY2018. Objective 1b. Protein/Oil. We continue to study the genetics of new alleles affecting protein and oil levels in soybean seeds. We are currently phenotyping populations for bulk segregant mapping. One problem that we have found is that protein content is a quantitative trait, and in our mapping cross populations we see evidence of small- effect QTL segregating in the population (we observe the same phenomenon with oleic acid.) To address this problem this year we are experimenting with using one of several alternative parents in the our mapping crosses, focused on soybean NAM population parents for which there is already a significant amount of genomic information. Objective 2 � Determine epigenetic patterns, modify gene expression. This year we finalized a manuscript describing the gene expression during soybean leaf senescence and it has been published online, and the dataset was made available in the Gene Expression Omnibus (GEO). (This is described in �Accomplishment 1�). Objective 3 - Evaluate the outcrossing and diversity of Phytophthora sojae populations and host plant interactions. In addition to Phytophthora sojae, another pathogen, P. sansomeana, also causes Phytophthora root rot of soybean. The type strain of this pathogen, 1819B, was obtained from a publicaly available collection. Its identity was confirmed by morphological observation and amplification and sequencing of the internal transcribed spacer region of the ribosomal RNA gene. The genome of this strain is being sequenced using the next-generation sequencing technologies. Objective 4 - Evaluate the population dynamics of Fusarium virguliforme. A world-wide collection of over 80 isolates of F. virguliforme from major soybean production regions where Soybean Death Syndrome(SDS) is a serious threat were assembled by ARS scientists in West Lafayette, Indiana. DNA was extracted from these isolates. Their identity was confirmed by PCR using species-specific primers. A bioinformatics pipeline was built to identify informative microsatellite loci by comparing genome assemblies of four strains. A total of 29 loci were identified. Experimental confirmation of bioinformatics predictions is in progress. Objective 5 - Organize, manage, and conduct the Northern Uniform Soybean Tests. The ARS scientists in West Lafayette, Indiana organized the 2016 Northern Uniform Soybean Tests and is organizing the 2017 tests. In 2016 tests, 397 soybean breeding lines and checks in maturity groups 00 to IV were evaluated for agronomic performance and disease resistance by 20 research groups at 50 sites in 11 states in the United States and 2 provinces in Canada. These breeding lines were submitted by public breeders in the Unites States and Canada. The ARS scientists in West Lafayette, Indiana directly evaluated lines in two sites in Indiana, and evaluated all lines for resistance to Phytophthora root rot under greenhouse conditions. The ARS scientists in West Lafayette, Indiana collected data from participants, analyzed the data, and published the results in the book �THE UNIFORM SOYBEAN TESTS NORTHERN REGION 2016�. The hard copy was delivered to participants and interested stake holders. The electronic copy of the book is freely available online. The 2017 tests are underway and a total of 407 public breeding lines and checks are being evaluated. In addition to agronomic traits and disease resistance, the 2017 tests also include evaluation of quality traits, such as fatty acid composition, protein contents, etc. Accomplishments 01 Probing the source-sink relationship in soybeans. The source-sink relationship is crucial for plant development and yield. While many studies have investigated the effect of leaves (source) on seed (sink) formation, the influence of the reproductive sink on source organs is largely unexplored. ARS researchers in West Lafayette, Indiana profiled the genes expressed in soybean leaves from plants that did not form seed tissues to look for markers of sink development and to determine how the biochemistry of these leaves differed from those on plants with normal seed formation. Preventing seed formation significantly delays the onset of leaf death. This research is an important part of the study of leaf aging and several genes and pathways were identified that could serve as future targets for modification to alter soybean leaf senescence and potentially impact yield. These findings will primarily be used by other scientists to understand gene function and alter the rate of leaf senescence in soybean. 02 Publication of the book �THE UNIFORM SOYBEAN TESTS NORTHERN REGION 2016�. The ARS lab in West Lafayette, Indiana organize the Northern Uniform Soybean Tests yearly. The tests evaluate soybean breeding lines for agronomic performance and disease resistance in northern states in the United States and provinces in Canada. The soybean lines are developed by public breeders in the United States and Canada that are suitable for planting in these regions. In addition to performing tests in multiple sites in Indiana, the ARS lab in West Lafayette, Indiana collected data from participants, analyzed the data, and published the 2016 test results in this book. The hard copy was delivered to participants and interested stake holders. The electronic copy of the book is freely available online. This book is used as primary evidence by breeders when making the decision to further advance their lines or release their lines to the public.
Impacts
(N/A)
Publications
- Sweeney, D., Carrero-Colon, M., Hudson, K.A. 2017. Characterization of new allelic combinations for high-oleic soybeans. Crop Science. 57(2):611-616.
- Brown, A., Hudson, K.A. 2017. Transcriptional profiling of mechanically and genetically sink-limited soybeans. Plant Cell and Environment. doi:10. 1111/pce.13030.
- Mantooth, K., Hadziabdic, D., Boggess, S., Windham, M., Miller, S., Cai, G. , Spatafora, J., Zhang, N., Staton, M., Ownley, B., Trigiano, R. 2017. Confirmation of independent introductions of an exotic plant pathogen of Cornus species,�Discula destructiva,�on the east and west coasts of North America and subsequent population bottlenecks. PLoS One. 12(7): e0180345. doi:10.1371/journal.pone.0180345.
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Progress 10/01/15 to 09/30/16
Outputs
Progress Report Objectives (from AD-416): 1) Identify genetic loci which contribute positive alleles to seed yield and quality traits and which control variation in seed quality, especially seed oil, protein, carbohydrate content, and fatty acid composition of legume seeds; develop strategies to facilitate effective transfer of useful genes into cultivated legumes; 1a) Mapping and characterizing mutant alleles that contribute to variation in seed fatty acid composition; 1b) Discovery of the molecular identity of genes that influence overall protein or oil content by forward genetics approaches. 2) Determine epigenetic patterns in soybean seeds during development, assess relationships between chromatin state and gene expression, and develop approaches to modify gene expression in developing seeds; 2a) Genome-wide profiling of epigenetic state and gene expression in developing soybean seeds by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequence-based expression profiling (RNA- seq). 3) Evaluate the outcrossing and diversity of Phytophthora sojae populations and host plant interactions. 4) Evaluate the population dynamics of Fusarium virguliforme. 5) Organize, manage, and conduct the Northern Uniform Soybean Tests. Approach (from AD-416): The project uses molecular biological approaches to study the regulation of genes that are required for soybean seed development. Biochemical approaches are used to study the interactions and function of gene products that are important for the accumulation of storage proteins or fatty acids in seeds. Molecular and genetic tools are used to define genes and genetic intervals that contribute to seed protein and oil content in mutant plants. Objective 1a. Identification of genetic loci contributing to soybean fatty acid composition. We have continued to identify mutations in genes that affect soybean fatty acid biosynthesis, this year publishing reports on soybeans with reduced palmitic acid and soybeans with increased levels of oleic acid. This year we advanced several double and higher order mutant combinations that carry defects in multiple genes in the fatty acid biosynthetic pathway, and we are evaluating the environmental stability of these combined traits. We have examined, for example, our ability to combine the elevated stearic acid and reduced linolenic acid traits, and will submit a manuscript on this before the end of the calendar year. This fiscal year we will submit a publication describing newly identified alleles for low linolenic soybean. We have expanded our fatty acid phenotyping pipeline to keep pace with our genotyping-by-sequencing based mapping approaches. We are using this approach to map several unidentified loci that affect oleic acid levels and linolenic acid levels, as well as genes for some morphological and physiological traits. This year, we have mapped a gene that affects soybean internode length to chromosome 17, and we are refining the map position of this gene using molecular markers. We have several composition traits (elevated oleic acid, elevated palmitic acid) in the mapping pipeline. In a related subordinate project funded by the United Soybean Board, we have utilized a �TILLInG-by-sequencing� strategy to identify a novel mutation in the RAFFINOSE SYNTHASE3 gene. We are still working to streamline and improve this method, and apply it to other genes of interest to our project. In this case we have obtained a mutant allele which can be combined with other genes to reduce the levels of the indigestible raffinose and stachyose oligosaccharides in soybean seeds, which makes soybean meal more efficiently digested by non-ruminant animals and increases meal value. We are currently doing genetic crosses and further evaluations of the effect of this mutation on soybean seed composition. We are likely to submit a manuscript on this in FY2017. Objective 1b. Protein/Oil. We are continuing to study the genetics of our new high protein traits by performing and evaluating genetic crosses to determine the additivity / allelism of our mutations with known high protein genes, and phenotyping populations for mapping. Objective 2 � Determine epigenetic patterns our chromatin IP experiments. In a related subordinate project to understand the transcriptional regulation of soybean leaf senescence, we have completed a follow-up study based on last year�s work to identify the transcriptional and biochemical differences between soybean leaves that senesce and leaves where senescence is delayed. We identified several gene families, including two transcription factor subfamilies that are highly overrepresented in leaves that do not senesce, and a manuscript is currently being prepared for submission. Objective 3 - Evaluate the outcrossing and diversity of Phytophthora sojae populations and host plant interactions. A total of 598 isolates of Phytophthora sojae were obtained from soils collected throughout Indiana during the first 12 months of this project. Each isolate was pathotyped and tested for sensitivity to fungicides metalaxyl and mefenoxam and this test was repeated for any specimens for which there was conflicting data. In addition, DNA has been obtained from each isolate for determining genotype. Each isolate was screened using species- specific primers in traditional PCR. Each isolate was screened with SSR markers to assess overall genetic variation as well as geographic influences on this variation. A landscape genetics approach was utilized to account for soil type, geography, planting dates, maturity groups and other variables that could lead to the diversification that is seen within the P. sojae populations. This data also revealed, while some populations are clonal as would be expected in a fungus, a high number of these populations are sexually reproductive based on levels of heterogeneity. A manuscript is ready for submission. Objective 5 - Organize, manage, and conduct the Northern Uniform Soybean Tests. Nearly 500 soybean breeding lines in maturity groups 00 to IV were submitted by public breeders in the U.S. and Canada for evaluation in 2015. Along with University and ARS scientists, lines were evaluated for agronomic performance and disease development in 18 field locations throughout the U.S. and Canada including 3 under the direction of ARS, West Lafayette, IN. In collaboration with plant pathologists at The Ohio State University and the University of Illinois at Urbana-Champaign, entries were evaluated under greenhouse conditions for resistance to the soybean cyst nematode and for race-specific and partial resistance to Phytophthora root rot (PRR). Data collected will be used by breeders to determine if a line is to be advanced, dropped, or released as a new variety for public and private use. The 2016 Northern Uniform Soybean Test data is available both online and in hard copy format. Accomplishments 01 Successful implementation of reverse genetics in soybean. While gene sequencing and RNA sequencing technologies have become routine and inexpensive, we still have many unanswered questions about how the genes function, and simply knowing a gene�s function cannot translate to practical utilization (breeding better soybeans) without a way to easily identify genetic variants. ARS researchers in West Lafayette, Indiana and collaborators implemented a method to use high throughput DNA sequencing to identify variants in any gene from large soybean mutant populations. A non-functional variant of a gene involved in carbohydrate biosynthesis in soybean seeds was identified. The elimination of this gene results in seeds with lower raffinose family oligosaccharide content, which makes a more efficient soybean meal. This variant is being evaluated in genetic crosses for its impact on carbohydrate composition, and will be useful to soybean breeders to improve soybean meal.
Impacts
(N/A)
Publications
- Thapa, R., Carrero-Colon, M., Crowe, M.D., Gaskin, E.L., Hudson, K.A. 2015. Novel FAD2-1A alleles confer an elevated oleic acid phenotype in soybean seeds. Crop Science. 56:226-231.
- Thapa, R., Carrero-Colon, M., Hudson, K.A. 2016. New alleles of FATB-1A to reduce palmitic acid levels in soybean. Crop Science. 56:1-5.
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Progress 10/01/14 to 09/30/15
Outputs
Progress Report Objectives (from AD-416): 1) Identify genetic loci which contribute positive alleles to seed yield and quality traits and which control variation in seed quality, especially seed oil, protein, carbohydrate content, and fatty acid composition of legume seeds; develop strategies to facilitate effective transfer of useful genes into cultivated legumes; 1a) Mapping and characterizing mutant alleles that contribute to variation in seed fatty acid composition; 1b) Discovery of the molecular identity of genes that influence overall protein or oil content by forward genetics approaches. 2) Determine epigenetic patterns in soybean seeds during development, assess relationships between chromatin state and gene expression, and develop approaches to modify gene expression in developing seeds; 2a) Genome-wide profiling of epigenetic state and gene expression in developing soybean seeds by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequence-based expression profiling (RNA- seq). Approach (from AD-416): The project uses molecular biological approaches to study the regulation of genes that are required for soybean seed development. Biochemical approaches are used to study the interactions and function of gene products that are important for the accumulation of storage proteins or fatty acids in seeds. Molecular and genetic tools are used to define genes and genetic intervals that contribute to seed protein and oil content in mutant plants. Objective 1. Identification of genetic loci contributing to soybean fatty acid composition. This year we have invested significant effort into rapid advancement of double and higher-order combinations of genes that we identified in previous years to improve oil content, including increased levels of oleic acid, decreased levels of linolenic acid, and increased levels of palmitic or stearic acid. We are analyzing progeny of these combinations as well as developing and thoroughly testing molecular markers that soybean breeders or geneticists can use to follow these genes within populations. We anticipate that these stacked traits will be more useful to breeders than individual mutations alone. We are studying lines that carry unknown mutations that result in alterations of the normal fatty acid profile. We have analyzed whole- genome resequencing data, and identified candidate polymorphisms to test for their association with mutant trait. In parallel we are using a bulk- segregant genotyping-by-sequencing (GBS) approach in these lines to quickly narrow in on the genomic region that contains the causative mutation, and expect to have preliminary map positions for ten genes (with different phenotypes) by the end of FY2015. Some of these lines have morphological phenotypes that can be scored visually in the field (for example, short internodes, or extra leaflets) in order to more quickly develop the genetic mapping method. Objective 2. We have made considerable progress this year to focus our efforts on our best lines with the most reproducible phenotypes for elevated seed protein or oil levels, and because we expanded our screen to include more mutants in previous years, we have many additional mutants to follow. For the 2015 crossing season, we have 15 lines that show an increase in protein content (ranging from 8-22% higher protein) or oil content (up to 10% higher than the wild type) planted in the field for crossing. We are also examining the ration of protein to oil in our mutant lines. We are collecting additional data in the field on these plants including overall morphology, seed size and number, and taking measurements of leaf photosynthesis. We have phenotyped two mapping populations from previous seasons that we are currently performing preliminary mapping on, including the testing of sequencing-based approaches to obtain map positions (as described in Objective 1). Objective 3. Determine epigenetic patterns during soybean development. We continue to analyze data from these experiments, and are performing additional replications to validate our findings. In a related subordinate project to understand the control of gene expression in soybean cotyledons and leaves, we have completed our gene expression analysis and have identified the expression patterns for the major groups of soybean transcription factors, which is documented in a recent publication (described below). Accomplishments 01 Developmental regulation of soybean genes during leaf senescence. The natural aging process of soybean leaves at the end of the season impacts their efficiency and the remobilization of nutrients (sugars, minerals and nitrogen) into seeds. To understand how soybean genes work together over time to control leaf aging and nutrient remobilization, ARS scientists at West Lafayette, IN studied gene expression in leaves and cotyledons of soybean. Genes were identified that were involved in the establishment of photosynthetic machinery or in the final processes of mobilization of nutrients. An important result of this experiment was defining the link between transcriptional regulators (control genes) and the specific sequence elements that they recognize in the control regions of the genes that they activate or repress during the later stages of leaf development. This data improves scientists� understanding of soybean gene function, and also helps identify how genes are regulated (particularly during the later stages of leaf development). These discoveries will allow more targeted biotechnology approaches by plant geneticists to modify leaf development and gene expression.
Impacts
(N/A)
Publications
- Brown, A.V., Hudson, K.A. 2015. Developmental profiling of gene expression in soybean trifoliate leaves and cotyledons. Biomed Central (BMC) Plant Biology. 15:169.
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Progress 10/01/13 to 09/30/14
Outputs
Progress Report Objectives (from AD-416): 1) Identify genetic loci which contribute positive alleles to seed yield and quality traits and which control variation in seed quality, especially seed oil, protein, carbohydrate content, and fatty acid composition of legume seeds; develop strategies to facilitate effective transfer of useful genes into cultivated legumes; 1a) Mapping and characterizing mutant alleles that contribute to variation in seed fatty acid composition; 1b) Discovery of the molecular identity of genes that influence overall protein or oil content by forward genetics approaches. 2) Determine epigenetic patterns in soybean seeds during development, assess relationships between chromatin state and gene expression, and develop approaches to modify gene expression in developing seeds; 2a) Genome-wide profiling of epigenetic state and gene expression in developing soybean seeds by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequence-based expression profiling (RNA- seq). Approach (from AD-416): The project uses molecular biological approaches to study the regulation of genes that are required for soybean seed development. Biochemical approaches are used to study the interactions and function of gene products that are important for the accumulation of storage proteins or fatty acids in seeds. Molecular and genetic tools are used to define genes and genetic intervals that contribute to seed protein and oil content in mutant plants. Objective 1 � Identification of genetic loci contributing to variation in seed fatty acid composition. We continue to work with soybean composition mutants isolated from our mutant population. This season, one of the main goals of our project is to develop lines that combine the best alleles to achieve improved fatty acid composition. We are making crosses and selecting lines which carry genes for increased oleic acid content and increased stearic acid content, increased oleic content and reduced linolenic acid content, and increased stearic acid content and reduced linolenic acid content. For each novel allele, we develop molecular markers to follow the gene in the population. These markers will make it easier for soybean breeders to use these genes in their own breeding programs. This year we initiated several projects to use genomic and sequence-based approaches to more rapidly identify the causative genes in our fatty acid mutant lines, as well as to use reverse-genetics based screening approaches to identify mutants within our population that may carry desirable composition traits. Of the five high oleic lines that we planned to study, the causative gene has been identified in two of the five already, and whole genomes will be resequenced for two of the remaining lines this year. We will perform bulk-segregant mapping for the high linolenate lines to identify map positions for causative genes. Objective 2 � Identification of genetic loci contributing to soybean seed protein content. This year we are working with several lines with genetically heritable changes in levels of soybean seed storage proteins, and initiated mapping of genes within our mapping populations that influence seed protein content in soybean. Using our mutagenized population, and following genes that result in either low or high protein levels in soybean seeds represents a novel approach to understanding the genetic control of seed protein levels. Objective 3 � Determine epigenetic patterns during soybean development. This year we re-assessed data from multiple stages of soybean seed development to determine the optimal stages for our study. Accomplishments 01 New sources of genetic variation for the high-oleic soybean oil trait. Soybean oil that is high in oleic acid content is healthier than conventional soybean oil in food products, and will thus result in a higher value soybean seed crop for producers. There are several soybean lines that have this trait, but new variants, and particularly non- transgenic variants, are likely to provide added value for the soybean oil market. Using a genetics approach, ARS researchers in West Lafayette, Indiana identified eight new variants of the most significant gene that controls soybean oleic acid content. These mutations alone result in a 20%- 50% increase in soybean seed oleic acid. Further, researchers designed and tested specific molecular markers that can be used to follow these genes in breeding populations, and used these markers to validate that these genes are associated with and causative for the high oleic acid trait in these new soybean lines. The impact of this work for farmers will be felt in a few years, after soybean breeding approaches incorporate these genes into elite soybean varieties that are widely grown, and stacked with other beneficial traits to improve the overall seed composition profile.
Impacts
(N/A)
Publications
- Carrero-Colon, M., Abshire, N., Sweeney, D., Gaskin, E., Hudson, K.A. 2014. Mutations in SACPD-C result in a range of elevated stearic acid concentration in soybean seed. PLoS One. 9(5): e97891. DOI:10.1371/journal. pone.0097891.
- Hudson, K.A., Hudson, M. 2014. The basic helix-loop-helix transcription factor family in the sacred lotus, Nelumbo nucifera. Tropical Plant Biology. 7(2):65-70.
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Progress 10/01/12 to 09/30/13
Outputs
Progress Report Objectives (from AD-416): 1) Identify genetic loci which contribute positive alleles to seed yield and quality traits and which control variation in seed quality, especially seed oil, protein, carbohydrate content, and fatty acid composition of legume seeds; develop strategies to facilitate effective transfer of useful genes into cultivated legumes; 1a) Mapping and characterizing mutant alleles that contribute to variation in seed fatty acid composition; 1b) Discovery of the molecular identity of genes that influence overall protein or oil content by forward genetics approaches. 2) Determine epigenetic patterns in soybean seeds during development, assess relationships between chromatin state and gene expression, and develop approaches to modify gene expression in developing seeds; 2a) Genome-wide profiling of epigenetic state and gene expression in developing soybean seeds by chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequence-based expression profiling (RNA- seq). Approach (from AD-416): The project uses molecular biological approaches to study the regulation of genes that are required for soybean seed development. Biochemical approaches are used to study the interactions and function of gene products that are important for the accumulation of storage proteins or fatty acids in seeds. Molecular and genetic tools are used to define genes and genetic intervals that contribute to seed protein and oil content in mutant plants. We are working with a number of soybean lines with altered fatty acid composition to characterize the genes involved in seed composition as well as to evaluate the effects of combination of mutant alleles to stack composition traits. We have identified 6 mutations in the SACPD-C gene and verified that these mutations are related to increased levels of stearic acid in soybean seeds. These mutations confer an increased level of stearic acid in the seed which will make soybean oil more useful in cooking fats. Another goal for the improvement of soybean oil is to increase the levels of oleic acid to increase the stability of the oil. During this project period we continued to characterize soybean fatty acid mutants with increased levels of oleic acid, identifying 7 lines carrying new mutations in known genes that result in high levels of oleic acid, and range in oleic acid content from 33-46%. These mutations may be combined to result in even higher levels of oleic acid and prove to be a useful resource for soybean breeders and geneticists. To identify novel mutations, we have expanded our efforts to generate both conventional and next-generation genetic mapping populations to identify the genes responsible for several other fatty acid traits, including alterations in linolenic acid, palmitic acid, and stearic acid. One milestone this year was to use the bulk segregant resequencing technique to identify genes responsible for altered fatty acid content. This technique uses next-generation sequencing on individuals from a segregating population that share a fatty acid phenotype to quickly identify linked genes. This growing season we have obtained the tissue and will finish the phenotyping of an additional generation to ensure that the lines that will be used for sequencing are true-breeding. We have initiated a new project to identify the transcriptional regulators bound to the promoters of seed storage protein genes during seed development, seed samples have been collected that will be analyzed in the fall.
Impacts
(N/A)
Publications
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