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United States Department of Agriculture

Agricultural Research Service

Research Project: FUNCTIONAL GENOMICS FOR IMPROVING NUTRIENTS AND QUALITY IN ALFALFA AND SOYBEAN
2013 Annual Report


1a.Objectives (from AD-416):
Objective 1: Use discoveries from Medicago truncatula, a model legume, root and root nodule genomics to characterize and improve the biological efficiency of symbiotic nitrogen (N2) fixation in alfalfa. Objective 2: Develop and employ RNA interference (i) -mediated gene silencing to identify the functional role of genes involved in phosphorus and nitrogen acquisition and metabolism in root tissues of legumes such as common bean, pea, and lentil. Objective 3: Determine genes regulating protein and oil accumulation in soybean through whole genome transcript analysis and functionally characterize gene candidates.


1b.Approach (from AD-416):
Alfalfa genes important in nitrogen fixation will be identified from the Medicago EST and genome sequencing projects. The sequence of 5'-promoter regions of selected genes will be identified from both alfalfa and M. truncatula. The expression of selected root nodule genes will be silenced through RNA interference. Nodules from plants containing RNA interference will be characterized for nitrogen fixation and nodule development. The Medicago truncatula gene chip will be used to assess global gene expression in alfalfa. BSL-1; Recertified May 19, 2011.


3.Progress Report:
This project terminated in FY13. To establish a genomics platform for alfalfa, transcript profiling on stems of two genotypes differing in cellulose and lignin concentration was done and used to create the first alfalfa gene index containing 124,025 unique sequences. A total of 1,294 simple sequence repeats, 10,826 single nucleotide polymorphisms and numerous allelic variations were identified. Candidate genes that may play a role in stem development and genes that may contribute to differences in cell wall composition in the two genotypes were identified. These genes can be used to improve alfalfa as a forage crop and cellulosic feedstock.

Phosphorus (Pi) is one of the most limiting nutrients for plant growth and development. However, the mechanisms for acclimation to Pi deficiency remain largely unknown. White lupin has unique adaptations for growth in Pi-deficient soils. RNA-sequencing technology was used to assess global gene expression in white lupin in response to Pi and assemble the first white lupin gene index. A total of 2,128 RNAs were differentially expressed in response to Pi deficiency. Twelve sequences were consistently differentially expressed due to Pi deficiency in Arabidopsis, potato and white lupin, making them ideal candidates to monitor Pi status of plants. This gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in acclimation to Pi deficiency and potential genes to use in improving plant acclimation to phosphorus deficiency.

The first gene expression atlas of soybean was developed from 14 diverse soybean tissues. Dramatic tissue-specific gene expression was found for the most highly expressed genes and for legume-specific genes in seed development and from root nodules. More than 177 genes were found to be involved in seed filling. The atlas provides a means of evaluating gene models for the soybean genome, provides a complete overview of genes expressed in soybean and lays a foundation for understanding genetic control of seed development. It also provides an in-depth view of genes expressed in nitrogen fixing root nodules.

A quantitative trait locus (QTL) on soybean chromosome 20, designated LG I, has a striking effect on seed protein content. A pair of soybean near-isogenic lines (NILs) contrasting in seed protein and differing in the genome segment containing the QTL were used to demarcate the QTL region to less than 5.4 million base pairs of DNA. Thirteen candidate genes were identified that mapped to the QTL and are being used to improve soybean seed quality.

Fast neutron (FN) radiation was used to induce deletions in the soybean genome to develop over 23,000 independent mutant lines. Variation for seed composition, maturity, morphology, pigmentation and nodulation traits was cataloged. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. A subset of 300 mutants was characterized, revealing gene deletion regions and candidate genes associated with phenotypes of interest. The FN mutant soybean population is a free public resource for soybean breeding and improvement.


4.Accomplishments
1. Understanding genetic regulation of soybean growth and development. A major objective for the community of scientists involved in soybean improvement is to develop genomic tools to understand soybean growth, development, and seed quality. In order to understand genetic regulation of important traits in soybean, ARS researchers at Saint Paul, Minnesota, in collaboration with University of Minnesota colleagues, developed a large population of soybean mutants and cataloged variation in seed composition, plant maturity, morphology, pigmentation and nodulation traits. Mutants that showed significant increases or decreases in seed protein and oil content across multiple generations and environments were identified. A subset of 300 mutants was characterized, revealing gene deletion regions and candidate genes associated with traits of interest. The mutant population has been made available as a free public resource for soybean breeding and improvement.

2. Phosphorus stress responsive genes for crop improvement. Although phosphorus (P) is abundant in soils, it is largely unavailable for uptake by plants and is frequently the most limiting nutrient for plant growth and development. Developing crop plants that are more efficient at acquiring and using P is critical for world agriculture. To identify plant genes responsive to and regulating P acquisition and use, ARS researchers in Saint Paul, Minnesota, in collaboration with University of Minnesota cooperators, found that sugars are involved in phosphorus stress signaling, and they identified a sucrose transporter (SUC2) that was highly expressed under phosphorus stress. A mutation in an unusual location in the Arabidopsis SUC2 gene results in very robust plant growth. Arabidopsis and soybean expressing the mutated SUC2 gene show enhanced growth and seed set. Directed mutagenesis of SUC2 may provide a new strategy for improving crop plant growth and development.

3. Increased nitrogen fixation in soybean. Specific organic acids (small carbon-containing compounds) have been implicated in improving symbiotic nitrogen fixation and plant nitrogen accumulation. ARS researchers in Saint Paul, Minnesota, in collaboration with University of Minnesota cooperators, developed two soybean lines that over-express the gene malate dehydrogenase (MDH) for malic acid production. One of the lines showed increased total nitrogen accumulation as compared to control plants. Over-production of specific forms of MDH appears to be an approach to improve nitrogen fixation and assimilation in legume plants. Improvement of nitrogen fixation and assimilation could reduce the need for nitrogen fertilizers and reduce production costs.

4. Development of legume gene atlases. Whole genome overviews of genes expressed in legume plants is valuable for identifying genes controlling many aspects of plant growth and development. ARS scientists in Saint Paul, Minnesota, conducted an analysis of the entire transcriptome (complete set of active genes) of four legume species: common bean, alfalfa, lupin, and soybean. For each species the results were organized into a gene expression atlas, which lists all of the genes active in a specific part of the plant such as leaves, roots, flowers, and seeds and the level of expression of each gene. A web-based graphical interface was developed by ARS and University of Minnesota collaborators to facilitate comparisons of expressed genes. This information is an invaluable resource for legume scientists for identifying genes controlling important traits such as protein nutritional quality and seed oil content and improving plant tolerance to changing environmental conditions.


Review Publications
O'Rourke, J.A., Iniguez, L.P., Bucciarelli, B., Roessler, J., Schumtz, J., McClean, P., Jackson, S.A., Hernandez, G., Graham, M.A., Stupar, R.M., Vance, C.P. 2013. A re-sequencing based assessment of genomic heterogeneity and fast neutron-induced deletions in a common bean cultivar. Frontiers in Plant Genetics and Genomics. DOI:10.3389/fpls.2013.00210.

Stec, A.O., Bhaskar, P.B., Bolon, Y., Nolan, R.K., Shoemaker, R.C., Vance, C.P., Stupar, R.M. 2013. Genomic heterogeneity and structural variation in soybean near isogenic lines. Frontiers in Plant Genetics and Genomics. DOI:10.3389/fpls.2013.00104.

Last Modified: 10/21/2014
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