SOYBEAN MOLECULAR VARIATION AND PROSPECTS FOR IMPROVEMENT

• Sponsoring Institution: State Agricultural Experiment Station
• Project Status: COMPLETE
• Funding Source: STATE
• Reporting Frequency: Annual
• Accession No.: 0215061
• Project Start Date: Jul 1, 2011
• Project End Date: Jun 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIV OF MINNESOTA
(N/A)
ST PAUL,MN 55108

Performing Department
Agronomy & Plant Genetics

Non Technical Summary
Soybean is one of the most important crops in the world. In recent years, the soybean community has committed abundant resources towards the development of technologies to sequence the soybean genome and assess DNA sequence diversity among different soybean varieties, with the hope of eventually using this information to target the improvement of soybean traits. My primary research area, which is greatly enabled by the genome sequence resource, focuses on the impact of nucleotide and structural genomic variation on phenotypic diversity in soybean traits. I am interested in the impact of both natural (within-species) and induced (fast neutron mutant) genetic variation. We are currently developing an extensive data set that can be used for investigating the relationship between gene deletions and soybean trait variation. My group maintains a particular interest in the function of gene duplicates within soybean, a paleopolyploid species in which ~75% of all genes are duplicated. We are using transcription analyses and developing a suite of site-directed mutations to understand the function of gene duplicates in the soybean RNAi pathway, which is a pathway essential for viral defense and genome stability. Lastly, we are initiating a project to elucidate soybean centromere function. This is the first step towards developing a soybean haploid-inducer line, which may someday be useful for soybean doubled haploid breeding. Doubled haploid technologies vastly accelerate the development of elite inbred lines in a breeding program.

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	20%
201	1820	1080	20%
202	1820	1040	20%
202	1820	1080	20%
206	1820	1040	10%
206	1820	1080	10%

Knowledge Area
206 - Basic Plant Biology; 202 - Plant Genetic Resources; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1820 - Soybean;

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

Keywords

soybean

fast neutron

structural variation

copy number variation

gene duplication

polyploidy

centromere

cenh3

doubled-haploid

Goals / Objectives
1. Identify soybean structural genomic variation and assess implications for soybean phenotypic variation. 2. Perform physical and genetic mapping of induced genome modifications in a soybean fast neutron mutant population. 3. Assess transcriptional, genetic and functional variation of soybean homoeologous genes. 4. Delineate soybean centromere function and assess potential for developing soybean haploid-inducer lines. 5. Provide molecular genetic support for applied programs.

Project Methods
Several emerging molecular methods are allowing us to make several new discoveries about gene content and structural variation within natural and mutated soybean lines. These methods are diagnostic in that they allow us to identify the subset of genes that vary among soybean accessions and mutant lines. Furthermore, the emerging site-directed nuclease technologies allows us to specifically target new variation within genes of interest. 1) Comparative Genomic Hybridization (CGH). Rapid and affordable genomic structural variation assessments may be obtained by using microarray-based Comparative Genomic Hybridization (CGH) platforms. The microarray hybridization procedure requires that we isolate genomic DNA from two samples: a reference accession (oftentimes cultivar Williams 82) and an accession of interest (AOI). The two samples are respectively labeled with either Cy5 or Cy3 dyes and co-hybridized to our CGH microarray. The relative intensity of the AOI to the reference accession will provide an approximation of relative deletions or additions of the two accessions to one another throughout the twenty soybean chromosomes. 2) Exome resequencing. In this procedure, DNA from an AOI is hybridized to a microarray that only contains probes designed from known soybean exon regions. The hybridized DNA is thereby captured on the microarray, and can then be isolated for resequencing analyses. The resequencing data can be aligned and compared with the reference sequence of cultivar Williams 82. The presence/absence of nearly all the genes in the genome can be assessed using this data. Additionally, this data can be used to identify point mutations within the respective genomes. 3) Genome resequencing. The most direct way to compare soybean genomes is through direct resequencing of the full genome for a given accession and then comparing to the published reference sequence of cultivar Williams 82. The cost of high-throughput resequencing has dropped dramatically in recent years, making this a more viable option. 4) Site-directed mutagenesis. We are using zinc-finger nuclease (ZFN) TAL effector nucleases (TALEN) transgene constructs to target several soybean genes. Whole-plant transformation using these methods generates new mutants within the gene of interest, but are not typically genetically linked to the nuclease transgene. Therefore, in subsequent generations it is possible to generate offspring that carry the stable mutation(s) but do not carry the original transgene that served to create the mutation.

Progress 07/01/11 to 06/30/16

Outputs
Target Audience:Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Between Jan-June, 2016, four lab personnel presented posters at the Plant and Animal Genome Conference: Ben Campbell (grad student), Shaun Curtin (post-doc), Jun Liu(post-doc) and Justin Anderson (post-doc). How have the results been disseminated to communities of interest?Five peer-reviewed publications and one review resulted from this project in 2016. The PI presented one professional talk between Jan and June, 2016. Stupar regularly presents his work at grower functions, including the Minnesota Ag Expo. What do you plan to do during the next reporting period to accomplish the goals?This is the final report for this project.

Impacts
What was accomplished under these goals? The cultivated soybean germplasm is known to have a relatively narrow genetic base, as compared with other crop species. This project is aimed at increasing our understanding of soybean genetic diversity and developing novel genetic resources to broaden the germplasm. We have used novel methodologies to map deletion and duplication mutations affecting soybean seed composition and architecture, and to investigate genomic differences within and among soybean cultivars. Recent discoveries have identified mutations and genes that underlie morphological and seed composition traits. Recent examples include the identification of a genes critical for chlorophyll biosynthesis, trichome development, and seed oil and sucrose composition (Campbell et al. 2015, 2016). We used environmental and genetic information to identify genetic regions that may confer abiotic stress resistance in wild soybeans, the genes of which can be bred into domesticated soybean (Anderson et al. 2015). Furthermore, we have demonstrated the relative variation in genome structure of different germplasm sources, such as transgenic and mutagenized plants (Anderson et al. 2016). Lastly, we are developing site-directed mutagenesis methods for soybean targeting seed composition and nutrient transporter traits; advanced were made in the past year on utilizing the CRISPR/Cas9 system for this goal (see Michno et al. 2015; Curtin et al. 2015).

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Campbell BW, Hofstad AN, Sreekanta S, Fu F, Kono TJY, ORourke JA, Vance CP, Muehlbauer GJ, Stupar RM. (2016) Fast Neutron induced structural rearrangements at a soybean NAP1 locus result in gnarled trichomes. Theor Appl Genet 129:1725-1738.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Anderson JE, Michno JM, Kono TJY, Stec AO, Campbell BW, Curtin SJ, Stupar RM. (2016) Genomic variation and DNA repair associated with soybean transgenesis: A comparison to cultivars and mutagenized plants. BMC Biotechnol 16:41.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Kono TJY, Fu F, Mohammadi M, Hoffman PJ, Liu C, Stupar RM, Smith KP, Tiffin P, Fay JC, Morrell PL. (2016) The role of deleterious substitutions in crop genomes. Mol Biol Evol 33:2307-2317.
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Campbell BW, Stupar RM. (2016) Soybean Mutant and Germplasm Resources: Current Status and Future Prospects. Curr Protoc Plant Biol 1:307-327. doi: 10.1002/cppb.20015
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Anderson JE, Kono TJ, Stupar RM, Kantar MB, Morrell PL. (2016) Environmental Association Analyses Identify Candidates for Abiotic Stress Tolerance in Glycine soja, the Wild Progenitor of Cultivated Soybeans. G3 (Bethesda) 4: 835-843.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Curtin SJ, Michno JM, Campbell BW, Gil-Humanes J, Mathioni SM, Hammond R, Gutierrez-Gonzalez JJ, Donohue RC, Kantar MB, Eamens AL, Meyers BC, Voytas DF, Stupar RM. (2015) microRNA Maturation and microRNA Target Gene Expression Regulation Are Severely Disrupted in Soybean dicer-like1 Double Mutants. G3 (Bethesda) 6: 423-433.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Graduate students have attended several professional conferences and presented posters. For example, student Benjamin Campbell presented a poster of his work at the Plant and Animal Genome conference and won first prize in his category. Campbell was also awarded the National Council of Commercial Plant Breeders Graduate Student Award for 2015. Master's student co-advisee Austin Dobbels was awarded a United Soybean Board Graduate Fellowship to pursue his PhD studies with soybean breeder Aaron Lorenz. Graduate students Ben Campbell and Jean-Michel Michno attended and gave oral presentations at the Soybean Mutant Finder event and Precision Genomics workshop. How have the results been disseminated to communities of interest?Two peer-reviewed publications resulted from this project in the reporting period. The PI presented invited seminars at six scientific conferences, three regional events and two University symposia. The PI regularly presents his work at grower functions, including the annual Minnesota Soybean Symposium. What do you plan to do during the next reporting period to accomplish the goals?We will advance our research aims by continuing current projects. We are on the verge of identfying several different mutant alleles that underlie soybean seed composition, architecture, and other morphological traits. We will continue to develop the CRISPR/Cas9 system for targeted mutagenesis in soybean, particularly for seed composition traits.

Impacts
What was accomplished under these goals? The cultivated soybean germplasm is known to have a relatively narrow genetic base, as compared with other crop species. This project is aimed at increasing our understanding of soybean genetic diversity and developing novel genetic resources to broaden the germplasm. We have used novel methodologies to map deletion and duplication mutations affecting soybean seed composition and architecture, and to investigate genomic differences within and among soybean cultivars. Recent discoveries have identified novel chromosomal phenomena following radiation mutagenesis, including a prevalence of segmental duplications on chromosome ends. We have identified causative nucleotide variants that underlie a suite of different traits; the first such paper was published this year on a chlorotic mutant phenotype and we have two more papers in preparation for other traits of interest. We are developing site-directed mutagenesis methods for soybean targeting seed composition and nutrient transporter traits; advanced were made in the past year on utilizing the CRISPR/Cas9 system for this goal (see Michno et al. publication).

Publications

• Type: Journal Articles Status: Published Year Published: 2014 Citation: Campbell BW, Mani D, Curtin SJ, Slattery RA, Michno JM, Ort DR, Schaus PJ, Palmer RG, Orf JH, Stupar RM (2015) Identical substitutions in magnesium chelatase paralogs result in chlorophyll-deficient soybean mutants. G3 (Bethesda) 5: 123-131.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Michno JM, Wang X, Liu J, Curtin SJ, Kono TJ, Stupar RM. (2015) CRISPR/Cas mutagenesis of soybean and Medicago truncatula using a new web-tool and a modified Cas9 enzyme. GM Crops Food (in press).

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate students have attended several professional conferences and presented posters. For example, student Benjamin Campbell presented a poster of his work at the ASA-CSSA-SSSA conference and won first prize in his category. Campbell also gave an oral presentation at the 15th Biennial Molecular & Cellular Biology of the Soybean Conference. Several personnel (Austin Dobbels, Ben Campbell, Jean-Michel Michno) attended and gave oral presentations at the Soybean Mutant Finder event and Precision Genomics workshop. How have the results been disseminated to communities of interest? Three peer-reviewed publications and one review resulted from this project in 2014. The PI presented three professional talks in the US and three in China. Stupar regularly presents his work at grower functions, including the Minnesota Ag Expo. What do you plan to do during the next reporting period to accomplish the goals? We will advance our research aims by continuing current projects. We have nearly identified an important mutation underlying a branching architecture trait. Genes residing within regions of structural variation will be tested for specific gene functions in response to stresses. We will continue to develop the CRISPR/Cas9 system for targeted mutagenesis in soybean, particularly for seed composition traits.

Impacts
What was accomplished under these goals? The cultivated soybean germplasm is known to have a relatively narrow genetic base, as compared with other crop species. This project is aimed at increasing our understanding of soybean genetic diversity and developing novel genetic resources to broaden the germplasm. We have used novel methodologies to map deletion and duplication mutations affecting soybean seed composition and architecture, and to investigate genomic differences within and among soybean cultivars. Recent discoveries have identified novel chromosomal phenomena following radiation mutagenesis, including a prevalence of segmental duplications on chromosome ends. We have used the genetic mapping tools to isolate genes responsible for soybean photosynthesis and trichome development. We are developing site-directed mutagenesis methods for soybean targeting seed composition and nutrient transporter traits.

Publications

• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Tek AL, Stupar RM, Nagaki K (2015) Modification of centromere structure: a promising approach for haploid line production in plant breeding. Turk J Agric For (in press).
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Bolon YT, Stec AO, Michno JM, Roessler J, Bhaskar PB, Ries L, Dobbels AA, Campbell BW, Young NP, Anderson JE, Grant DM, Orf JH, Naeve SL, Muehlbauer GJ, Vance CP, Stupar RM (2014) Genome resilience and prevalence of segmental duplications following fast neutron irradiation of soybean. Genetics 198: 967-981.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Anderson JE, Kantar MB, Kono TY, Fu F, Stec AO, Song Q, Cregan PB, Specht JE, Diers BW, Cannon SB, McHale LK, Stupar RM (2014) A Roadmap for Functional Structural Variants in the Soybean Genome. G3 (Bethesda) 4: 1307-1318.
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Stupar RM (2014) To the reference and beyond: understanding variation in plant genomes. Brief Funct Genomics 13: 255-256.

Progress 01/01/13 to 09/30/13

Outputs
Target Audience: Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Graduate student Justin Anderson presented a poster of his work at the American Seed Trade Association meeting (winning first prize in his category). Post-doc Yung-Tsi Bolon presented a poster of her work at the Cold Spring Harbor laboratories. Post-doc Fengli Fu presented a post of her work at the U of MN MPGI Science on the Spot event. Several personnel (Justin Anderson, Ben Campbell, Jean-Michel Michno, Yung-Tsi Bolon) attended and gave oral presentations at the Soybean Mutant Finder event and Precision Genomics workshop. How have the results been disseminated to communities of interest? Three peer-reviewed publications resulted from this project in 2013. The PI presented eight professional talks in the US and one in South Korea. Stupar regularly presents his work at grower functions, including the Minnesota Ag Expo. What do you plan to do during the next reporting period to accomplish the goals? We will advance our research aims by continuing current projects. Genes lying within regions of structural variation will be tested for specific gene functions in response to stresses. We will test the CRISPR/Cas9 system for targeted mutagenesis in soybean, particularly for seed composition traits.

Impacts
What was accomplished under these goals? The cultivated soybean germplasm is known to have a relatively narrow genetic base, as compared with other crop species. This project is aimed at increasing our understanding of soybean genetic diversity and developing novel genetic resources to broaden the germplasm. Research platforms have been developed to detect large deletions and duplications among different soybean lines. We have used these methods to map mutations affecting soybean seed composition and architecture and to investigate genomic differences within and among soybean cultivars. Recent discoveries have identified an enrichment of natural deletions and duplications with gene clusters encoding plant defense response signal transduction pathways, providing insight into the mechanisms of soybean resistance gene evolution. We have developed site-directed mutants for soybean targeting the post-transcriptional gene silencing and nutrient transporter pathways. We are currently developing methodologies to modify gene clusters arranged as tandem repeats to enhance soybean resistance to pathogens.

Publications

• Type: Journal Articles Status: Published Year Published: 2013 Citation: Stec AO, Bhaskar PB, Bolon YT, Nolan R, Shoemaker RC, Vance CP, Stupar RM (2013). Genomic heterogeneity and structural variation in soybean near isogenic lines. Front Plant Sci 4: 104. Stupar RM, Specht JE (2013) Insights from the Soybean (Glycine max and G. soja) Genome: Past, Present and Future. Adv Agron 118: 177204. Curtin SJ, Anderson JE, Starker CG, Baltes NJ, Mani D, Voytas DF, Stupar RM (2013) Targeted Mutagenesis for Functional Analysis of Gene Duplication in Legumes. Methods in Molc Biol 1069: 25-42.

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

Outputs
OUTPUTS: Talks: Presented seminars detailing our work at the following venues: 1) USDA-ARS Sclerotinia Initiative Annual Meeting, Bloomington, MN. Jan 2012; 2) Plant and Animal Genome XX Conference, NimbleGen workshop, San Diego, CA. Jan 2012; 3) VI International Conference on Legume Genetics and Genomics, Next Generation Genomics session, Hyderabad, India. Oct 2012. Published abstracts accompanied all the presentations. PARTICIPANTS: Personnel: Robert Stupar (Assistant Professor/Project Leader), Jean-Michel Michno (Junior Scientist and Lab Manager), Adrian Stec (Assistant Scientist), Theresa Brenberg (Junior Scientist), Ryan Donohue (Junior Scientist), Yung-Tsi Bolon (Post-doc), Fengli Fu (Post-doc), Justin Anderson (Graduate Student), Benjamin Campbell (Graduate Student) Cooperators: Carroll Vance, ARS, University of Minnesota; James Orf and Daniel Voytas, University of Minnesota; Leah McHale (Ohio State University); Scott Jackson, University of Georgia; Randy Shoemaker, ARS, Iowa State University TARGET AUDIENCES: Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
The cultivated soybean germplasm is known to have a relatively narrow genetic base, as compared with other crop species. This project is aimed at increasing our understanding of soybean genetic diversity and developing novel genetic resources to broaden the germplasm. Research platforms have been developed to detect large deletions and duplications among different soybean lines. We have used these methods to map mutations affecting soybean seed composition and architecture and to investigate genomic differences within and among soybean cultivars. Recent discoveries have identified an enrichment of natural deletions and duplications with gene clusters encoding plant defense response signal transduction pathways, providing insight into the mechanisms of soybean resistance gene evolution. We have developed site-directed mutants for soybean targeting the post-transcriptional gene silencing and nutrient transporter pathways. We are currently developing methodologies to modify gene clusters arranged as tandem repeats to enhance soybean resistance to pathogens.

Publications

• McHale LK, Haun WJ, Xu WW, Bhaskar PB, Anderson JE, Hyten DL, Gerhardt DJ, Jeddeloh JA, Stupar RM (2012) Structural variants in the soybean genome localize to clusters of biotic stress response genes. Plant Physiol 159: 1295-1308.
• Curtin SJ, Voytas DF, Stupar RM (2012) Genome Engineering of Crops with Designer Nucleases. Plant Genome 5: 42-50.
• Kantar M, Betts K, Hulke BS, Stupar RM, Wyse D (2012) Breaking Tuber Dormancy in Helianthus tuberosus L. and Interspecific Hybrids of Helianthus annuus L. x Helianthus tuberosus. Hort Sci 47:1342-1346.
• Curtin SJ, Kantar MB, Yoon HW, Whaley AM, Schlueter JA, Stupar RM (2012) Co-expression of soybean Dicer-like genes in response to stress and development. Funct Integr Genomics 12: 671-682.

Progress 01/01/11 to 12/31/11

Outputs
OUTPUTS: Talks: Presented seminars detailing our work at the following venues: 1) Plant and Animal Genome XVIII Conference, NimbleGen Workshop, San Diego, CA. Jan, 2011; 2) X-GEN Congress and Expo, Mapping and Assembly session, San Diego, CA. Mar 2011; 3) XIV National Congress of Biochemistry and Plant Molecular Biology and 7th Symposium Mexico-USA, Campeche, Mexico. Dec 2011. Published abstracts accompanied all the presentations. PARTICIPANTS: Personnel: Robert Stupar (Assistant Professor/Project Leader), Jean-Michel Michno (Junior Scientist and Lab Manager), Adrian Stec (Assistant Scientist), Shaun Curtin (Post-doc), Pudota B. Bhaskar (Post-doc, Justin Anderson (Graduate Student), Benjamin Campbell (Graduate Student), Dhananjay Mani (Graduate Student) Cooperators: Carroll Vance, ARS, University of Minnesota; James Orf and Daniel Voytas, University of Minnesota; Leah McHale (Ohio State University); Simon Chan (University of California - Davis); Scott Jackson, University of Georgia; Randy Shoemaker, ARS, Iowa State University TARGET AUDIENCES: Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has resulted in the development of methodologies for understanding the transcriptional and functional differentiation of duplicated genes. Transcriptional and site-directed mutagenesis methodologies targeted at duplicate gene pairs have recently been published. We have developed genomic methodologies for detecting deletions and copy number variants between different soybean lines. We have used these methods to map mutations affecting soybean seed composition and architecture and to investigate genomic differences within and among soybean culivars. We have developed site-directed mutants for soybean targeting the post-transcriptional gene silencing and nutrient transporter pathways.

Publications

• Curtin SJ, Zhang F, Sander JD, Haun WJ, Starker C, Baltes NJ, Reyon D, Dahlborg EJ, Goodwin MJ, Coffman AP, Dobbs D, Joung JK, Voytas DF, Stupar RM (2011) Targeted mutagenesis of duplicated genes in soybean with zinc finger nucleases. Plant Physiol 156: 466-473.
• Bolon YT, Haun WJ, Xu WW, Grant D, Stacey MG, Nelson RT, Gerhardt DJ, Jeddeloh JA, Stacey G, Muehlbauer GJ, Orf JH, Naeve SL, Stupar RM, Vance CP. (2011) Phenotypic and genomic analyses of a fast neutron mutant population resource in soybean. Plant Physiol 156: 240-253.
• Haun WJ, Hyten DL, Xu WW, Gerhardt DJ, Albert TJ, Richmond T, Jeddeloh JA, Jia G, Springer NM, Vance CP, Stupar RM (2011) The composition and origins of genomic variation among individuals of the soybean reference cultivar Williams 82. Plant Physiol 155: 646-655.
• Sander JD, Dahlborg EJ, Goodwin MJ, Cade L, et al. (2011) Selection-free zinc-finger-nuclease engineering by context-dependent assembly (CoDA). Nat Methods 8: 67-69.

Progress 01/01/10 to 12/31/10

Outputs
OUTPUTS: Talks: Presented seminars detailing our work at the following venues: 1) Plant and Animal Genome XVIII Conference, Legumes Workshop, San Diego, CA, Jan, 2010; 2) Soybean Research & Technology Transfer Annual Overview Session, Shakopee, MN, Feb 2010; 3) 2010 Plant Breeding Symposium, Plant Breeding: The Genomics and Post-Genomics Era, University of Minnesota, St. Paul, MN, Mar 2010; 4) Purdue University, Agronomy and Soil Science departmental seminar, April 2010.; 5) Strategic Advances in Soybean Genomics Research Workshop, St. Louis, MO, July 2010; 6) The 13th Biennial Molecular & Cellular Biology of the Soybean Conference, Session I: Whole Genome Analysis: Focus on Technology, Durham, NC, Aug 2010; 7) 2010 Microbial and Plant Genomics Institute Symposium: Biological Applications of Next-Generation Sequencing, University of Minnesota, St. Paul, MN, Sept 2010; 8) ASA-CSSA-SSSA 2010 International Annual Meetings, Session: Green Revolution 2 through Application of Second Generation Sequencing to Plant Breeding and Improving Quantitative Traits, Long Beach, CA, Nov 2010. Published abstracts accompanied presentations 1, 3, 6, 7, 8. PARTICIPANTS: Personnel: Robert Stupar (Assistant Professor/Project Leader), Andrew Coffman (Junior Scientist and Lab Manager), Shaun Curtin (Post-doc), Dhananjay Mani (Graduate Student). Cooperators: Carroll Vance, ARS, University of Minnesota; James Orf and Daniel Voytas, University of Minnesota; Jer-Young Lin and Scott Jackson, Purdue University, West LaFayette, IN; Randy Shoemaker, ARS, Iowa State University, David Hyten, ARS, Beltsville, MD. TARGET AUDIENCES: Legume genomicists, geneticists and breeders; evolutionary biologists; users and producers of soybean. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has resulted in the development of methodologies for understanding the transcriptional and functional differentiation of duplicated genes. We have applied the Sequenom MassARRAY technology to measure transcriptional divergence of soybean homoeologous genes and identify novel deletion mutants between homoeolog pairs. We have developed genomic methodologies for detecting deletions and copy number variants between different soybean lines. We have used these methods to map mutations affecting soybean seed composition (and other traits) and to investigate genomic differences within and among soybean culivars. We have developed site-directed mutants for soybean targeting the post-transcriptional gene silencing and nutrient transporter pathways.

Publications

• Stupar RM (2010) Into the wild: The soybean genome meets its undomesticated relative. Proc Natl Acad Sci, USA (in press).
• Lin JY, Stupar RM, Hans C, Hyten DL, Jackson SA (2010) Structural and functional divergence of a 1-MMb duplicated region in the soybean (Glycine max) genome and comparison to an orthologous region from Phaseolus vulgaris. Plant Cell 22: 2545-2561.
• Severin AJ, Peiffer G, Xu WW, Hyten DL, Bucciarelli B, O'Rourke JA, Bolon YT, Grant D, Farmer AD, May GD, Vance CP, Shoemaker RC, Stupar RM (2010) An integrative approach to genomic introgression mapping. Plant Physiol. 154: 3-12.
• Gardiner SA, Boddu J, Berthiller F, Hametner C, Stupar RM, Adam G, Muehlbauer GJ (2010) Transcriptome analysis of the barley-deoxynivalenol interaction: evidence for a role of glutathione in deoxynivalenol detoxification. Mol Plant Microbe Interact. 23: 962-976.

Progress 01/01/09 to 12/31/09

Outputs
OUTPUTS: Talks: Presented the seminar "Transcriptional Divergence Of Homoeologous Soybean Genes On Linkage Groups A2 And E" at the Plant & Animal Genome XVII Conference, San Diego, CA, Jan. 2009. Presented the seminar "Transcription profiling and mutation detection of soybean homoeologous genes" at the World Soybean Research Conference VIII, Beijing, China, Aug. 2009. Published abstracts accompanied both presentations. PARTICIPANTS: Personnel: Robert Stupar (Assistant Professor/Project Leader), Andrew Coffman (Junior Scientist and Lab Manager), Shaun Curtin (Post-doc), Dhananjay Mani (Graduate Student). Cooperators: Carroll Vance, ARS, University of Minnesota; James Orf, University of Minnesota; Jer-Young Lin and Scott Jackson, Purdue University, West LaFayette, IN. TARGET AUDIENCES: Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has resulted in the development of methodologies for understanding the transcriptional and functional differentiation of duplicated genes. We have applied the Sequenom MassARRAY technology to measure transcriptional divergence of soybean homoeologous genes and identify novel deletion mutants between homoeolog pairs. We are developing genomic methodologies for detecting deletions and copy number variants between different soybean lines. We are using genomic methodologies to identify genes responsible for soybean abiotic stress variation. We are in the process of identifying the members of gene families responsible for soybean floral induction and post-transcriptional gene silencing.

Publications

• Chaudhary B, Flagel LE, Stupar RM, Udall JA, Verma N, Springer NM, Wendel J. (2009) Reciprocal Silencing, Transcriptional Bias and Functional Divergence of Homeologs in Polyploid Cotton (Gossypium). Genetics 182: 503-517.

Progress 01/01/08 to 12/31/08

Outputs
OUTPUTS: Talks: Presented seminar for the Minnesota Agriculture Experiment Station Report, Feb 2008. Presented the seminars "Molecular crop improvement" and "Current projects" to the Minnesota Soybean Research & Promotion Council, Sept 2008. Presented the seminar "Transcriptional divergence and subfunctionalization of homoeologous soybean genes" at the IV International Conference on Legume Genomics and Genetics, Satellite Workshop on Phaseolae Genomics: soybean-common bean interface, Puerto Vallarta, Mexico, Dec. 2008. Presented the poster "Structural variation and transcriptional subfunctionalization of Dicer-like genes in soybean" at the IV International Conference on Legume Genomics and Genetics, Dec. 2008. PARTICIPANTS: Personnel: Robert Stupar (Assistant Professor/Project Leader), Andrew Coffman (Junior Scientist and Lab Manager), Shaun Curtin (Post-doc), Dhananjay Mani (Graduate Student) Cooperators: Jer-Young Lin and Scott Jackson, Purdue University, West LaFayette, IN TARGET AUDIENCES: Legume genomicists, geneticists and breeders; evolutionary biologists. Users and producers of soybean. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
This project has resulted in the development of methodologies for understanding the transcriptional and functional differentiation of duplicated genes. We have applied the Sequenom MassARRAY technology to measure the transcriptional divergence of 30 linked gene duplicates in soybean, demonstrating that the duplicates exhibit high rates of transcriptional subfunctionalization while maintaining conserved express profiles among related genotypes. We are in the process of identifying the members of gene families responsible for soybean floral induction and post-transcriptional gene silencing.

Publications

• No publications reported this period