SOYBEAN GENOME MAPPING AND GENETIC MECHANISMS OF ABIOTIC STRESS TOLERANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0210348
• Project No.: MO-PSSL0660
• Project Start Date: Jun 1, 2007
• Project End Date: May 31, 2012

Project Director
Nguyen, HE, T..

Recipient Organization
UNIVERSITY OF MISSOURI
(N/A)
COLUMBIA,MO 65211

Performing Department
Plant Sciences

Non Technical Summary
Among the different abiotic stresses, drought and waterlogging are the important stresses limiting soybean production. Drought is the most important one, reduces soybean yield about 40%. Water is a scarce resource and its avalability limit soybean production. Our main purposes are to identify QTLs/genes tolerance to drought or waterlogging in soybean. Subsequently, these genes/QTLs will be used for molecular breeding purposes via marker assisted selection (MAS) for the development of drought and waterlogging tolerant soybean lines.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
201	1820	1040	50%
203	1820	1080	50%

Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

Field Of Science
1040 - Molecular biology; 1080 - Genetics;

Keywords

soybean

qtl

genetic map

physical map

abiotic stress

drought

waterlogging

gene expression

proteomics

Goals / Objectives
Objective 1: To develop a high density genetic map and integration of genetic map into physical map of soybean. (a) Construction of high density genetic linkage map of soybean: A good genetic map provides a scaffold for studies of soybean structural genomics and genetic analysis of genes related to important agronomic traits. The soybean integrated genetic map is well developed, but the size of mapping populations is too small to map genes in a fine scale. In order to construct a high resolution of genetic map and minimize the segregation distortion of markers, we initially used 760 F2 lines derived from the cross Forrest x Williams82 for construction of framework map using polymorphic SSR markers. (b) Integration of genetic map into physical map of soybean: A standard sequence-ready physical map from Williams82 was identified as a prerequisite for many advanced genomic studies. We have created a six-dimensional pool of BAC clones representing 5-fold redundancy of the soybean genome. We intend to use a BAC pooling strategy to place novel loci (via SSR-based PCR) onto the physical map. These novel markers will come from sequences not previously mapped via hybridization to BAC-end sequences or by Overgo probes. These will ensure that additional, novel markers are placed onto the physical map. Moreover, the development and use of the BAC pools will provide a general mapping resource for the community. Objective 2: To investigate the genetic and molecular basis of abiotic stress tolerance in soybean. (a) Molecular mapping of abiotic stress (drought and waterlogging) tolerance in soybean: Plants possess several adaptive and constitutive traits to endure during period of drought. In soybean, rooting depth, water use efficiency, nitrogen fixation, leaf wilting, and osmotic adjustment are the most important traits to evaluate for drought tolerance. To investigate the genetic and molecular basis of abiotic stress tolerance, especially drought and waterlogging, different parents have been selected based on the traits of interest and development of mapping populations are in progress. (b) Functional genomics of drought tolerance in soybean: The contrasting growth and developmental responses of different soybean tissue types to water deficits provide important information for making molecular comparisons. The goal of this project is to have a better understanding of the molecular and genetic mechanisms that determine root and shoot growth response and the partitioning of assimilates into the major seed reserve components under water deficit conditions. Different functional genomics tools like microarray, proteomics, metabolomics and translational genomics will be used to investigate molecular basis of drought tolerance in soybean.

Project Methods
To develop a high density genetic map and integration of genetic map into physical map of soybean. (a) In order to construct a high resolution genetic map and minimize the segregation distortion of markers, we initially used 760 F2 lines derived from the cross Forrest x Williams82 for construction of framework map using SSR markers. Using the selective mapping strategy, we will select a core set of mapping population for subsequent SNP markers using high throughput platforms. A standard genetic map with full coverage of genome and high resolution will be constructed by integration of several RIL maps with the F2 map. (b) In order to integrate genetic map into physical map of soybean, we will contribute in anchoring 1500-2000 additional markers (e.g., SNP, SSRs) to the high resolution Williams82 physical map. We have created a six-dimensional pool of BAC clones representing 5-fold redundancy of the soybean genome. Additional SSR markers will come from our sequencing efforts and these SSR primers will be mapped and used against the pooled BAC libraries to identify individual BACs associated with each locus. We intend to use a BAC pooling strategy to place novel loci (via SSR-based PCR) onto the physical map. To investigate the genetic and molecular basis of soybean tolerance to abiotic stresses. (a) Molecular mapping of abiotic stress tolerance: In soybean, rooting depth, water use efficiency, nitrogen fixation, leaf wilting, and osmotic adjustment are the most important traits to evaluate for drought tolerance. Different parents have been selected based on the traits of interest and development of mapping populations are in progress. Genotyping will be done at the soybean genome mapping lab at MU. Parents and mapping population will be evaluated in the greenhouse and/or in the field for different traits related to drought tolerance. For waterlogging, recombinant inbred lines along with parents will be evaluated to determine the levels of tolerance in replicated plots under both flooded and non-flooded conditions at two locations in Missouri. Genotyping, genetic map construction and QTL analysis will be done following standard lab protocols. Currently, we have more than 1,000 SSR and 1,000 SNP markers available for QTL mapping. (b) Functional genomics of drought tolerance in soybean: For gene expression profiling, we will use soybean root and leaf under well water and water deficit conditions. Soybean seedling will be used to study how total protein composition changes in association with the differential growth responses of the root and shoot to water deficits among different genotypes. We will compare the seed development profiles to examine how drought stress influences transcription regulation and protein composition at different seed filling stages. The highly abundant proteins will be analyzed using peptide finger printing. The high throughput transcription factor (TF) profiling will be conducted to discover stress specific and seed developmental specific TFs.

Progress 06/01/07 to 05/31/12

Outputs
Target Audience:Ultimately soybean producres, especially in the Midwest regions will be the beneficiaries of this project. The outcome of this project will help to build a foundation for incorporating exotic drought tolerance traits into elite soybean lines and varities to minimize yield losse under stress conditions. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The overall aim of this project is to identify and characterize novel genes that control traits of interest through functional genomics and genome mapping research followed by the utilization of this information for development of improved cultivars for soybean producers. Eventhough there were no any FTE workingin thisproject, several postodcotoral fellows and undergraduate student assistants had an oppurtunity to learn and familiarize with with the areas of research including genetics, genomics and bioinformatics How have the results been disseminated to communities of interest?As par of the University Missoiur soybeanresearchser's database, the results and tools associated with this porject wasdessiminated to the communities inclduing researchers and farmers through the SoyKB, a web resources for soybean research. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. We have constructed a high density linkage map of soybean 2. We havbe developed a high resolution soybean genetic map with minimum segregation distortion of markers byutilizing alarge pouplation of recombinant lines. 3. We identified 3800 single feature polymorphism (SFP) markers using Affymetrix GeneChip, and ~200 have been validated. In addition, we are sequencing a bulked library of the small fragments derived from Williams 82 and Forrest genome by Solexa high throughput sequencing approach. This will lead to the identification of a large number of SNP or indel markers. 4. Succesfully ingtegrated thegentic map into teh physical map. BAC pooling and sequencing strategies were used to achieve this goal. 5. Idnetifed new germpalsm for better biotic and abiotic stres tolerance capaciteis and these parents were utilised in the gene/QTL dsicvoery associated with stress tolerance. 6.We have characterized transcripts and transcript profiles in regions of growth maintenance and inhibition in soybean roots under water deficit in comparison to well-watered conditions. The transcriptome characterization of apical and basal regions of soybean root growth zone under early (5h) and longer duration (48h) water deficits were conducted using soybean Affymetrix Genechip. Several transcripts belong to specific metabolic and regulatory pathways were identified in response to water stress. These results reveal that drought stress-responsive transcripts are largely root region specifi 7. Several mapping populations were developed to identify genes/genomic regions associated with abiotic stres s tolerance. The genetic mapping of new resistance genes and/or QTLs for SCN, drought, water logging and salinity, and discovery and development of new genetic markers such as single nucleotide polymorphisms (SNPs), mapping of candidate genes, and use of high-throughput genotyping program through MAS will promote breeding efficiency to develop genetic materials resistant to multiple races of SCN and different abiotic stress.

Publications

• Type: Book Chapters Status: Published Year Published: 2012 Citation: Manavalan, P.L. and H.T. Nguyen. 2012. Drought tolerance in crops: Physiology to genomics. In: Plant Stress Physiology. S. Shabala (ed.), CABI, UK.
• Type: Book Chapters Status: Published Year Published: 2009 Citation: Hanumappa, M., and H.T. Nguyen. 2009. Genetic approaches toward improving heat tolerance in plants. In: Genes for Plant Abiotic Stress. M. Jenks and A. Wood (eds.). Wiley-Blackwell, USA.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Le, D.T., D.L. Aldrich, B. Valliyodan, Y. Watanabe, C.V. Ha, R. Nishiyama, S.K. Guttikonda, T.N. Quach, J.J. Gutierrez-Gonzalez, L.S. Tran, and H.T. Nguyen. 2012. Evaluation of candidate reference genes for normalization of quantitative RT-PCR in soybean tissues under various abiotic stress conditions. PLoS One. 7(9):e46487.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Nguyen, T.H., L. Brechenmacher, J. Aldrich, T. Clauss, M. Gritsenko, K. Hixson, M. Libault, K. Tanaka, F. Yang, Q. Yao, L. Pasa-Tolic, D. Xu, H.T. Nguyen, and G. Stacey. 2012. Quantitative phosphoproteomic analysis of soybean root hair inoculated with Bradyrhizobium japonicum. Mol Cell Proteomics. 11:1140-1155.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Ha, J., B. Abernathy, W. Nelson, D. Grant, X. Wu, H.T. Nguyen, G. Stacey, Y. Yu, R.A. Wing, R.C. Shoemaker, and S.A.Jackson. 2012. Integration of the Draft Sequence and Physical Map as a Framework for Genomic Research in Soybean (Glycine max (L.) Merr.) and Wild Soybean (Glycine soja Sieb. and Zucc.). G3 (Bethesda) 2 (3):321-329.
• Type: Journal Articles Status: Published Year Published: 2011 Citation: Wu, X., T.D. Vuong, J.A. Leroy, J.G. Shannon, D.A. Sleper, and H.T. Nguyen. 2011. Selection of a core set of RILs from Forrest x Williams 82 to develop a framework map in soybean. Theor Appl Genet. 122:1179-1187.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: 52. Joshi, T., K. Patil, M.R. Fitzpatrick, L.D. Franklin, Q. Yao, J.R. Cook, Z. Wang, M. Libault, L. Brechenmacher, B. Valliyodan, X. Wu, J. Cheng, G. Stacey, H.T. Nguyen, and D. Xu. 2012. Soybean Knowledge Base (SoyKB): a web resource for soybean translational genomics. BMC Genomics. Suppl 1:S15
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Pathan, S., H. Nguyen, R. Sharp, and J.G. Shannon. 2010. Soybean improvement for drought salt and flooding tolerance. Korean J. Breed. Sci. 42(4): 329-338.
• Type: Journal Articles Status: Published Year Published: 2010 Citation: Wang, Z., M. Libault, T. Joshi, B. Valliyodan, H.T. Nguyen, D. Xu, G.Stacey, and J. Cheng. 2010. SoyDB: a knowledge database of soybean transcription factors. BMC Plant Biol. 10:14.

Progress 01/01/07 to 12/31/07

Outputs
OUTPUTS: The overall aim of this project is to identify and characterize novel genes that control traits of interest through functional genomics and genome mapping research followed by the utilization of this information for development of improved cultivars for soybean producers. As part of the first objective of this project, we have anchored all BAC contigs onto the soybean whole genome sequence assembly and that would enable us to use sequence resource to target candidate genes and marker development for marker assisted selection. We have mapped over 1550 of genetic markers to the physical map using 6D-BAC pool and in addition to this, Sequence Tagged Sites (STSs) derived from Expressed sequence tag (EST) associated with biotic and abiotic stress responses and seed composition traits are being mapped concurrently. We have constructed a framework genetic map with ~400 molecular markers using 760 F2 population and the construction of a Gold-standard high resolution genetic map for soybean is in progress. In the past year, extensive efforts were put on SNP discovery and validation for the STS markers via different approaches. We also identified 3800 single feature polymorphism (SFP) markers using Affymetrix GeneChip, and ~200 have been validated. In addition, we are sequencing a bulked library of the small fragments derived from Williams 82 and Forrest genome by Solexa high throughput sequencing approach. This will lead to the identification of a large number of SNP or indel markers. As part of the objective 2 of the project, development of RILs for drought tolerance mapping is in progress. We have characterized transcripts and transcript profiles in regions of growth maintenance and inhibition in soybean roots under water deficit in comparison to well-watered conditions. The transcriptome characterization of apical and basal regions of soybean root growth zone under early (5h) and longer duration (48h) water deficits were conducted using soybean Affymetrix Genechip. Several transcripts belong to specific metabolic and regulatory pathways were identified in response to water stress. These results reveal that drought stress-responsive transcripts are largely root region specific. We have constructed Full length cDNA library from the mixed stress root tissues (drought, waterlogging, salinity) and the sequencing efforts, both ESTs and selected full lengths are in progress in collaboration with DOE-JGI (Department of Energy Joint Genome Institute). Studies were conducted in the greenhouse to determine the flood tolerance mechanisms of the plant introduction PI408105A compared to the flood-sensitive genotype, S99-2281. The genotype PI408105A showed consistent tolerance to flooding as determined by overall plant injury. PI408105A plants more rapidly developed an adaptive mechanism to flooding by producing aerenchyma and adventitious roots and were able to resume root growth after three days of flooding in comparison to the flood sensitive genotype S99-2281. Roots of PI408105A plants also contained more ATP and showed less membrane damage than roots of S99-2281 plants. 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
Biotechnology tools that allow researchers to manipulate soybean at the molecular level and assist breeders in making selections based on genetic properties of soybean provides powerful technology to soybean breeding programs. Crop genetic improvement continues to be a challenging task for breeders as they try to incorporate several desirable traits in a final cultivar for release. These traits include higher yield, environmental stress tolerance, pathogen resistance especially soybean cyst nematode (SCN), and desirable levels of seed oil and protein composition. We have developed different mapping populations to identify genes and/or QTLs linked for resistance to drought and water logging tolerance. The genetic mapping of new resistance genes and/or QTLs for SCN, drought, water logging and salinity, and discovery and development of new genetic markers such as single nucleotide polymorphisms (SNPs), mapping of candidate genes, and use of high-throughput genotyping program through MAS will promote breeding efficiency to develop genetic materials resistant to multiple races of SCN and different abiotic stress.

Publications

• Nunberg, A., J.A. Bedell, M.A. Budiman, R.W. Citek, S.W. Clifton, L. Fulton, D. Pape, Z. Cai, T. Joshi, H.T. Nguyen, D. Xu, and G. Stacey. (2006). Survey sequencing of soybean elucidates the genome structure and composition. Functional Plant Biology 33: 1-9.
• Zhang, X.C., Wu, X., Findley, S., Wan, J., Libault, M., Nguyen, H.T., Cannon, S.B., Stacey, G. (2007). Molecular evolution of lysin motif-type receptor-like kinases in plants. Plant Physiol. 144:623-36.
• Shannon, J.G., J.A. Wrather, D.A. Sleper, H.T. Nguyen, and S.C. Anand. (2007). Registration of Stoddard soybean. J. of Crop Registrations 1: 28-29.
• Guttikonda, S., Valliyodan, B., Nguyen, H.T. 2007. Genetic engineering of AtDREB1D transcription factor to improve drought tolerance in soybean. Frontiers in Transgenesis, Danforth Center International Fall Symposium, St. Louis, MO.
• Neelakandan, A.K., Valliyodan, B., Nes, D.W., Nguyen, H.T. 2007. Bioengineering phytosterol accumulation in soybean. Missouri Life Sciences symposium, University of Missouri-Columbia, Missouri.
• Wu, X., G. Zhong, S. Findley, P. Cregan, G. Stacey, H.T. Nguyen. (2008). Genetic marker anchoring by six-dimensional pools for development of a soybean physical map. BMC Genomics 9:28 doi:10.1186/1471-2164-9-28.
• Shoemaker, R.C., Grant, D., Olson, T., Warren, W.C., Wing, R., Yu, Y., Kim, H., Cregan, P., Joseph, B., Futrell-Griggs, M., Nelson, W., Davito, J., Walker, J., Wallis, J., Kremitski, C., Scheer, D., Clifton, S.W., Graves, T., Nguyen, H., Wu, X., Luo, M., Dvorak, J., Nelson, R., Cannon, S., Tomkins, J., Schmutz, J., Stacey, G., Jackson, S. (2008). Microsatellite discovery from BAC end sequences and genetic mapping to anchor the soybean physical and genetic maps. Genome. 51:294-302.
• Flores, T., O. Karpova, X. Su, P. Zeng, K. Bilyeu, D.A. Sleper, H.T. Nguyen, Z.J. Zhang. (2008). Silencing of GmFAD3 gene by siRNA leads to low alpha-linolenic acids (18:3) of fad3-mutant phenotype in soybean [Glycine max (Merr.)]. Transgenic Res. 10.1007/s11248-008-9167-6
• Shannon, J.G., J.A. Wrather, D.A. Sleper, R.T. Robbins, H.T. Nguyen, and S. Anand. (2007). Registration of Jake soybean. J. of Crop Registrations 1: 29-30.
• Vuong, T.V., X. Wu, M.S. Pathan, B. Valliyodan, and H.T. Nguyen. 2007 Genomics approaches to soybean improvement. In. Genomics-assisted crop improvement. P. K. Varshney and R. Tuberosa (eds), Springer USA.
• Valliyodan, B., Libault, M., Xu, D., Stacey, G., Nguyen, H.T. 2007. Identification of abiotic stress specific molecular switches for stress tolerance and enhanced seed composition in soybean through high throughput quantitative real time PCR. Plant Biology 2007, American Society for Plant Biology, Chicago, IL.
• Wu, X., G. Zhong, S. Findley, P. Cregan, G. Stacey, H.T. Nguyen. (2008). Genetic marker anchoring by six-dimensional pools for development of a soybean physical map. BMC Genomics 9:28 doi:10.1186/1471-2164-9-28
• Lee, J-D., J-K. Yu., Y-H. Hwang., S. Blake., Y-S. So., G-J. Lee, H.T. Nguyen., and J. G. Shannon. (2008). Genetic diversity of wild soybean (Glycine soja Sieb. & Zucc) accessions from South Korea and other countries. Crop Science 48:606-616.
• Valliyodan, Babu and H.T. Nguyen. 2008. Genomics of abiotic stress in soybean. In: Soybean Genomics. Gary Stacey (ed.). Springer, USA. (in press)
• Pathan, M.S., J-D. Lee, J.G. Shannon, and H.T. Nguyen. 2008. Recent advances in breeding for drought and salt stress tolerance in soybean. In: Advances in molecular-breeding toward drought and salt tolerant crops. M. A. Jenks, P. M. Hasegawa, and S.M. Jain (eds), Springer USA. (in press)