August 12, 2012 Welcome

Twenty-six years ago the Biennial Cellular and Molecular Biology of the Soybean Conference began here in Iowa. Since then it has travelled to many states and institutions across the U.S. During this time, our research community has watched soybean cellular and genetic resources expand from the use of biochemical markers to the completion of the soybean genome sequence. With these resources, soybean research has flourished, advancing critical research and promoting soybean crop improvement.

We are pleased to welcome two plenary speakers. On Sunday evening, Dr. Carroll Vance will provide a historical perspective on soybean introduction and production in the U.S. In addition, Dr. Vance will highlight current and future research that leverages the soybean genome sequence to improve soybean yield and quality. During Monday morning’s plenary session, Dr. Jeff Doyle will address the adaptive success of legume polyploids and implications for crop improvement. The sessions that follow will include cutting edge research reports on pests and diseases, translation of genetic and genomic knowledge into soybean advancement, seed composition and nutrition, biotic interactions, functional genomics, genome structure and evolution and more.

As you know, this conference would not have been possible without the generous support of our sponsors. In particular, we would like to acknowledge the organizers of the 2010 soybean conference, the Crop Science Department of North Carolina State University, for supporting the travel of minority and underrepresented students and post-docs. We are delighted by the number of people taking advantage of this opportunity. In addition, we would like to recognize Jennifer Vit with Iowa State University’s Conference Planning and Management for her invaluable help in organizing the conference.

We are proud that the conference has returned to Iowa. We hope you enjoy the conference and your stay in Iowa.

Sincerely,

Randy C. Shoemaker, Chair Michelle Graham, Sponsorship Chair David Grant, Website Development and Abstract Chair Gustavo Macintosh, Travel Grant Chair Madan Bhattacharyya, Organizing Committee Steven Cannon, Organizing Committee Basil Nikolau, Organizing Committee Sponsor Acknowledgements

Our sponsors have helped make SOY 2012 a conference that promotes the exchange of research and ideas between scientists from around the world. Their generous funding has helped lower the cost of the meeting, allowing even more students and scientists to participate. Please join us in thanking our sponsors:

GOLD SPONSORS:

SILVER SPONSOR:

AGENDA

Sheraton West Des Moines

Sunday, August 12, 2012 Sponsored by DuPont Pioneer

3:00pm - Registration 6:30pm Atrium Set up Posters Polk and Dallas Rooms

6:15pm Welcome Ballrooms Randy Shoemaker, USDA-ARS and Iowa State University, Ames, Iowa

6:30pm DuPont Pioneer Plenary Address Carroll Vance, USDA-ARS and University of Minnesota, St. Paul, Minnesota From George Washington to the Genome: Leveraging Genetics and Molecular Biology to Improve Soybean

7:30pm Refreshments served (Heavy hors d’oeuvres) Atrium Posters open for viewing Polk and Dallas Rooms

8:30pm Dinner – On your own (see restaurant guide in abstract book)

9:00pm Informal Gathering Salon B/C (2nd Floor) Entertainment by the band SEVEN

Monday, August 13, 2012

Morning sessions sponsored by Monsanto All sessions take place in the Ballroom unless otherwise noted.

7:00am - Registration Open 10:00am Atrium

7:00am Continental Breakfast Atrium

8:15am Monsanto Plenary Address Jeff J. Doyle, Cornell University, Ithaca, New York From the first seed plant to the present (and future): the role of polyploidy in shaping genomes and phenotypes in soybean and its relatives

Morning Session I: Pests and Diseases Session Chairs: Leah McHale and Melissa Mitchum

9:00am Gustavo MacIntosh, Iowa State University Soybean aphid suppression of plant defenses: mechanisms and effects on other plant-pest interactions

9:20am Feng Qu, The Ohio State University Engineering RNA1-based resistance to viruses, insects, and other pathogens

9:40am Melissa Mitchum, University of Missouri Molecular underpinnings of incompatible SCN-soybean interactions

10:00am Refreshment Break Sponsored by Monsanto Atrium Posters open for viewing Dallas and Polk Rooms

Morning Session II: Pests and Diseases Session Chairs: Leah McHale and Melissa Mitchum

10:45am Andrew Bent, University of Wisconsin SCN resistance determinants at the Rhg1 locus

11:05am Yuan Chao Wang – Nanjing Agricultural University The facility of bioinformatics and discovery of new Avr effectors of Phytophthora sojae

11:25am Madan Bhattacharyya, Iowa State University Genetic analyses suggest that the FvTox1 toxin produced by Fusarium virguliforme is involved in foliar SDS development in soybean

11:45am Nilwala Abeysekara, Iowa State University Mapping quantitative trait loci encoding partial resistance to Phytophthora sojae in soybean

12:00pm Lunch Sponsored by Monsanto Atrium

Afternoon Session III: Translational Genomics Sponsored by Syngenta Session Chairs: Glenn Bowers, David Hyten and James Specht

1:30pm Kristin Bilyeu, USDA-ARS, Columbia, MO Applying technology to help resolve real-world issues: the case of developing low phytate/high available phosphate soybeans

1:50pm Wayne Parrot, University of Georgia Debugging Soybean

2:10pm Brian Diers, University of Illinois Fine mapping Rag1 and Rag2 and the evaluation of new aphid resistance sources

2:30pm Tom Clemente, University of Nebraska Transposition of the maize Ds element in the soybean genome

2:50pm Refreshment Break Sponsored by Syngenta Atrium Posters open for viewing Polk and Dallas Rooms

Afternoon Session IV: Translational Genomics Session Chairs: Glenn Bowers, David Hyten & James Specht

3:30pm Sally Mackenzie, University of Nebraska Breeding the epigenome in soybean

3:50pm Knut Meyer, DuPont Dissecting control of carbon partitioning in developing seeds by enhancer tagging: characterization of Arabidopsis mutants and translational work in soybeans

4:10pm James Specht, University of Nebraska Soybean yield potential and genome re-sequencing: stepping into the final frontier! (A last hurrah perspective from an old-timer)

4:30pm Anna Joe, University of Nebraska GRP7, a substrateof Pseudomonas syringae Type III Effector HopU1, plays a role in plant innate immunity

4:45pm Poster Sessions (light hors d’oeuvres) Sponsored by United Soybean Board & Iowa Soybean Association

6:00pm Dinner on your own (see restaurant guide in abstract book)

8:00pm Informal Gathering Salon B/ C (2nd Floor) Entertainment by the band SEVEN

Tuesday, August 14, 2012 All sessions take place in the Ballroom unless otherwise noted.

7:00am Continental Breakfast Atrium Posters open for viewing Polk and Dallas Rooms

Morning sessions sponsored by BASF Plant Science Morning Session V: Abiotic Stress Session Chairs: Jamie O’Rourke and Yung-Tsi Bolon

8:15am Jamie O’Rourke, USDA-ARS, University of Minnesota Investigating nitrogen deficiency in common bean

8:35am Jessica Schlueter, University of North Carolina-Charlotte Transcription analysis of ozone-response in susceptible and tolerant soybean lines

8:55am Michelle Graham, USDA-ARS, Iowa State University DNA replication and the iron deficiency response in soybean

9:15am Henry Nguyen, University of Missouri Exploitation of root system architecture for improving drought tolerance

9:35am Yuji Yamasaki, Indiana University and Purdue University Responsiveness of soybean CBF and COR genes to cold stress

9:50am Refreshment Break Sponsored by BASF Plant Science Atrium Posters open for viewing Dallas and Polk Rooms

Morning Session VI: Composition/Nutrition Session Chairs: Kristin Bilyeu and Tom Clemente

10:30am John Browse, Washington State University Making the best soybean oil that we can

10:50am Tim Durrett, Kansas State University Using a novel acyltransferase to alter the structure of plant triglycerides for targeted applications

11:10am Doug Allen, USDA-ARS/Donald Danforth Plant Science Center Analyzing developing soybean seed metabolism with isotopic labeling and metabolic flux analysis

11:30am Tony Kinney, DuPont Commercialization of high oleic soybeans

11:50am Sarah I. Jones, University of Illinois Using RNASeq to profile soybean seed development from fertilization to maturity

12:05 Lunch Sponsored by BASF Plant Science Atrium

Afternoon Session VII: Functional Genomics Session Chairs: Gustavo MacIntosh and Steve Whitham

1:30pm Marc Libault, University of Oklahoma Functional characterization of soybean transcription factors using comparative and genomic approaches

1:50pm Nathan Hancock, University of South Carolina-Aiken max mutation: mPing-based gene discovery

2:10pm Yung-Tsi Bolon, USDA-ARS, University of Minnesota How resilient is the soybean genome? Insights from fast neutron mutagenesis

2:30pm Steve Whitham, Iowa State University Discovery of gene networks regulating soybean defenses using virus- induced gene silencing

2:50pm Emily Pierce, University of Georgia Metabolic engineering of carotenoid production in soybean

3:05pm Refreshment Break Atrium Posters open for viewing Polk and Dallas Rooms

Afternoon Session VIII: Biotic Interactions I Session Chairs: Janine Sherrier and Gary Stacey

3:45pm Janine Sherrier, University of Delaware Small RNAs as regulators of nodulation

4:05pm Gary Stacey, University of Missouri Soybean root hairs: a single cell model for systems biology

4:25pm Russ Carlson, University of Georgia Glycoprofiling of the soybean root hair cell wall

4:45pm Posters open for viewing Polk and Dallas Rooms

5:30pm Transportation to the Iowa Historical Building Bus loads at north hotel entrance

6:00pm Reception with hors d’oeuvres and cash bar Self tour of The Iowa State Historical Museum

7:15pm Banquet with entertainment by Southern Reign Iowa State Historical Building Sponsored by United Soybean Board & Iowa Soybean Association

8:30pm - Busses return to Sheraton West Des Moines Hotel 9:30pm

Wednesday, August 15, 2012 All sessions take place in the Ballroom unless otherwise noted.

7:00am Continental Breakfast Atrium Posters open for viewing Dallas and Polk Rooms

Morning Session IX: Biotic Interactions II Session Chairs: Janine Sherrier and Gary Stacey

8:15am Stewart Smith, Novozymes Inc. Basic and applied understanding of signal molecules from rhizobia (Lipo-chitooligosaccharides) contributes to better crop production

8:35am Hongyan Zhu, University of Kentucky Molecular genetics of nodulation and nitrogen fixation specificity in soybean and Medicago

8:55am Swarup Roy Choudhury, Donald Danforth Plant Science Center Heterotrimeric G-proteins regulate soybean nodulation

Morning Session X: Genome Structure and Evolution Session Chairs: Jessica Schlueter, Bob Stupar and Steven Cannon

9:15am Suk-Ha Lee, Seoul National University Genome divergence in Glycine max and soja

9:25am Qijian Song, USDA-ARS, University of Maryland Structure of high-resolution haplotype blocks in the soybean genome

9:45am Katherine Espinosa, Iowa State University Allele switching with the soybean Aconitase-2 and Aconitase-4 loci

10:00am Refreshment Break Sponsored by Schillinger Genetics Atrium Posters open for viewing Dallas and Polk Rooms

10:40am Jungmin Ha, University of Georgia Genome structure variation in Glycine species

11:00am Ashley Egan, East Carolina University Evolution of a complex disease resistance gene cluster in soybeans and relatives

11:20am Jianxin Ma, Purdue University Population resequencing reveals evolutionary propensities of the soybean genome

11:40am Robert Stupar – University of Minnesota Exploring structural variation in the soybean genome

12:00pm Concluding Remarks Randy Shoemaker and Robert Stupar Remove Posters

From George Washington Carver to the Genome: Leveraging Genetics and Molecular Biology to Improve Soybean

Carroll Vance, USDA/ARS, Agronomy and Plant Genetics, University of Minnesota

In 1904, George Washington Carver studying the composition of soybeans concluded that they are a valuable source of protein and oil. He proposed that rotating soybeans with other crops would replenish the soil with nitrogen and minerals for two years. His findings brought soybeans into the mainstream of agriculture. Between 1910 and 1929 Charles V. Piper and William J. Morse published several agronomic papers on the ‘wonder crop’ soybean. At that time only 20 varieties of soybean were available in the U.S. Growers held the first soybean field days in 1920 in Indiana and formed the National Soybean Growers’ Association (renamed American Soybean Association in late 1925). In the 1920’s Henry Ford tossed some soybean to his engineers and said “You guys are supposed to be smart. You ought to be able to do something with them”. By 1935 Mr. Ford was using one bushel of beans for every car produced. From 1924- 1964 soybean yields increased from 11 to 24 bushels/acre. From 1965-2011 yields increased from 24-42 bushels/acre. Most of the advances in soybean yield through 2011 came from the efforts of plant breeders and agronomists, prior to the sequencing of the genome in 2010. In the future, how can we leverage the genome sequence of Glycine to improve yield and quality? From the First Seed Plant to the Present (and Future): The Role of Polyploidy In Shaping Genomes and Phenotypes in Soybean and its Relatives

Jeff Doyle, Cornell University

Soybean biologists have long been confronted with the polyploid nature of Glycine max, whose somatic chromosome number of 2n = 40 stands out among other phaseoloid legumes that mostly are 2n = 20 or 22 (e.g., Phaseolus, Vigna, Cajanus). That difference is due to a polyploid event that could be as recent as around 5 million years ago, and which occurred in the ancestor soybean shares with the ca. 30 perennial, mostly Australian members of Glycine. That putatively allopolyploid duplication, however, is not the only one that shaped genomes of Glycine species. It is now known that the ancestor of all seed plants experienced a whole genome duplication, as did the ancestor of all flowering plants. Furthermore, a later eudicot ancestor is thought to have undergone a genome triplication, and the ancestor of the largest lineage of legumes, the papilionoids, had yet another genome duplication. All of these events left their mark on the Glycine genome, gene families, and phenotypes, making understanding the process of polyploidy an important goal. The story of polyploidy in Glycine did not stop millions of years ago: it continues to be a significant phenomenon in the ongoing evolution of perennial Glycine species, having produced a cluster of species in the last 50,000 years some of which, unlike their 2n = 40 relatives, have spread from Australia to colonize islands of the Pacific Ocean. This kind of adaptability is often considered to be a hallmark of polyploids, and could result from such phenotypic advantages as higher photosynthetic rates or greater photoprotection. Finding the underlying explanations for the success of polyploids is a challenge with implications for both basic biology and agriculture. Soybean Aphid Suppression of Plant Defenses: Mechanisms and Effects on Other Plant-Pest Interactions

Gustavo MacIntosh, Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa

Aphids are specialized insects that feed on phloem sap and have a large impact on their plant hosts. They alter plant metabolism, affect plant growth and development, and also are vectors for many plant viruses. Due to their feeding habits, aphids produce little mechanical damage and avoid or deter many plant defenses commonly triggered by other herbivores. It has been proposed that aphids can suppress effective defenses through the induction of decoy responses. The soybean aphid is one of the main pests of soybean in the Midwest, causing yield reductions of up to 40% if left unattended. Important advances have been made in understanding the biology and ecology of the insect. However, the molecular mechanisms underlying plant susceptibility and resistance to this herbivore are still unknown.

Using transcriptome and metabolome analyses, we identified multiple phytohormone pathways that respond to aphid colonization. Our results showed that jasmonate (JA) signaling, a hormone response normally associated with defense against herbivores, is repressed in compatible interactions. At the same time, hormone signals associated with abiotic stress (abscisic acid, ABA) are induced, possibly as a decoy response. The repression of effective defenses seems to be exerted at different levels. Aphids cause a reduction in the levels of polyunsaturated fatty acids that are the substrate for JA biosynthesis, and also block the ability of the plant to respond to this hormone. Suppression of defenses seems to depend on the ability of aphids to trigger a decoy response coordinated by ABA. Plants in which the ABA response was silenced became more resistant to aphid colonization.

Manipulation of phytohormone pathways by aphids also results in altered response to other herbivores. Plants colonized by aphids are unable to deploy a full response to wounding, which could result in an increase in damage by defoliating insects. Aphid colonization also results in changes in phytohormone signaling in roots. As a consequence, aphid-infested plants are more susceptible to soybean cyst nematode infestations. The interaction between aphids and nematodes seems to be mediated by multiple pathways, including miRNA regulated phytohormone signals. Engineering RNAi-based Resistance to Viruses, Insects, and Other Pathogens

Feng Qu, Department of Plant Pathology, The Ohio State University, Columbus, Ohio

RNA interfence (RNAi) is a highly conserved genome surveillance system operating in most eukaryotic organisms to guard the cells against the invasion or proliferation of molecular parasites including transposons, transgenes, as well as viruses. Studies in recent years have demonstrated that RNAi-based resistance engineering is highly effective at achieving resistance against not only viruses, but also nematodes, insects, and fungal pathogens. We applied this strategy to soybean plants and observed robust resistance to multiple viruses with a single dsRNA-expressing transgene. Specifically, our transgene construct contained three short inverted repeats (IRs) with sequences of three soybean-infecting viruses (Alfalfa mosaic virus, Bean pod mottle virus, and Soybean mosaic virus). These IRs were assembled into a single transgene under control of the 35S promoter and terminator of Cauliflower mosaic virus. Three independent transgenic lines were obtained and all of them exhibited strong systemic resistance to the simultaneous infection of the three viruses in both greenhouse and field experiments. We are now testing the effectiveness of this strategy at controlling soybean aphids. Molecular Underpinnings of Incompatible SCN-soybean Interactions

Melissa Mitchum, University of Missouri

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, a microscopic roundworm that feeds on the roots of soybean is a major constraint to soybean production. This nematode causes more than $1 billion in yield losses annually in the United States, making it the most economically important pathogen on soybean. Despite the widespread deployment of resistant cultivars to manage this disease, the genes for resistance have not been cloned. Moreover, the increase in virulent populations of this parasite on most known resistance sources necessitates the development of novel approaches for control. Our current lack of understanding of the molecular basis of plant resistance and nematode virulence in this pathosystem continues to hinder progress to enhance the effectiveness and durability of natural plant resistance and enable the design of novel resistance strategies using biotechnological approaches. The first Rhg (for resistance to Heterodera glycines) genes were identified in the early 1960s. Since then, numerous reports are available on the identification and mapping of QTL in soybean underlying resistance to SCN from a variety of different germplasm sources. QTL on chromosomes 18 (rhg1) and 8 (Rhg4) are two major resistance QTL that have been consistently mapped in a variety of soybean germplasm. Plants carrying Rhg genes display an incompatible interaction between host and parasite. The plant’s roots are penetrated by infective juveniles, but feeding cells induced by the nematodes degenerate prematurely, causing them to die before reaching adult stages. We are interested in the network of molecular events that underlie incompatible SCN-soybean interactions. Laser capture microdissection coupled with transcript profiling has enabled the identification of soybean genes whose expression coincides with feeding cell degeneration in resistant cultivars. RNAi, VIGS, and overexpression screens are underway to evaluate the role of these genes in SCN resistance. A map-based cloning approach, coupled with newly developed functional genomic tools in soybean, has identified and confirmed the role of a gene at the Rhg4 locus in resistance to this pathogen. Surprisingly, the gene does not encode a protein belonging to one of the canonical classes of disease resistance proteins used by plants to defend against pathogens. These findings represent major breakthroughs in the field that will no doubt have immediate impact in the mechanistic understanding of the plant’s resistance against this parasite that can be readily exploited to improve nematode resistance of soybean, an increasingly important global crop. SCN Resistance Determinants at the Rhg1 Locus

Andrew Bent, Dept. of Plant Pathology, University of Wisconsin - Madison David Cook, Dept. of Plant Pathology, University of Wisconsin - Madison Xiaoli Guo, Dept. of Plant Pathology, University of Wisconsin - Madison Tong Geon Lee, Dept. of Crop Sciences, University of Illinois at Urbana-Champaign Sara Melito, Adam Bayless, Teresa Hughes, Jianping Wang, Myungsik Kim, Brian W. Diers, Matthew Hudson

Soybean cyst nematode (SCN, Heterodera glycines) is an extremely damaging pathogen of soybeans. Together with crop rotation, the PI88788-derived rhg1-b allele of the Rhg1 locus is the primary means by which SCN is managed in cultivated soybeans in the U.S. Previous genetic mapping defined an interval for rhg1-b that, in the SCN-susceptible but fully sequenced Williams 82 soybean genome, corresponds to a 67 kb interval carrying 11 predicted genes. Our work to identify the genetic features within this interval that contribute to SCN resistance will be presented. In transgenic roots derived from the SCN-resistant cultivar Fayette (which carries rhg1-b), resistance to SCN was significantly reduced when silencing constructs were used to target any of three genes: Glyma18g02580 (predicted amino acid transporter), Glyma18g02590 (predicted alpha-SNAP protein), or Glyma18g02610 (function unknown; wound-induced protein). No impacts on SCN were observed when other genes in the rhg1-b interval were silenced. We discovered that the above three genes are present in a ~30 kb block of genomic DNA that is present in multiple copies within the Rhg1 locus of PI88788- derived or Peking-derived soybeans, while this block is present in only one copy in tested varieties such as Williams 82 whose Rhg1 loci do not confer resistance to SCN. The very few encoded amino acid polymorphisms between these repeated blocks, and between the homologs from SCN-resistant vs. SCN-susceptible varieties, will be discussed. Strong gene expression polymorphisms are possibly as significant as amino acid polymorphisms, with greater constitutive expression of these three genes in the roots of resistant varieties. Differences in DNA methylation are also prominent between the alleles of these genes in resistant vs. susceptible varieties. Hence a combination of features within the Rhg1 locus contribute to Rhg1-mediated SCN resistance. The Facility Of Bioinformatics And Discovery Of New Avr Effectors Of Phytophthora sojae

Yuanchao Wang, Nanjing Agricultural University Suomeng Dong, Nanjing Agricultural University Weixiao Yin, Nanjing Agricultural University

Phytophthora sojae is a notorious oomycete pathogen producing a great loss on global soybean production annually. The disease outcome between soybean and P. sojae depends on whether hosts could recognize pathogen avirulence effectors. Recently identified oomycete avirulence effectors are characterized by N-terminal host targeting motif (RxLR motif), sequence and transcriptional polymorphisms between virulent and avirulent strains. Benefit from 454 genome sequencing and solexa transcriptome sequencing of P. sojae strains, eight RxLR effectors are bioinformatically identified based on their sequence and transcriptional polymorphism pattern. Genetic mapping work suggested that one of them, Avh307, perfectly matched Avr3b phenotype, another one Avh6 matched Avr1d phenotype. Transient expression of the ORF from avirulence strain on soybean specifically triggered Rps3b or Rps1d mediated program cell death, respectively, confirming it encodes avirulence effector Avr3b and Avr1d. Transient expression of Avr3b on Nicotiana benthamiana uncovered that the effector facilitates Phytophthora infection on and inhibits PCD induced by a variety of Phytophthora PAMPs, suggesting Avr3b avirulence effectors contribute to pathogenicity. Biochemistry assay further uncovered that Avr3b is an ADP-ribose pyrophosphorylase with ADPR and NADH as its preferable substrate. Thus, we assumed that the role of P. sojae avirulence effectors Avr3b is to suppress host immunity by interrupting ADPR and NAHD cycling. Genetic Analyses Suggest that the FvTox1 Toxin Produced by Fusarium virguliforme is Involved in Foliar SDS Development in Soybean

Madan Bhattacharyya, Department of Agronomy, Iowa State University, Ames, Iowa Hargeet Brar, Sivakumar Swaminathan, Ramesh Pudake, Jordan L. Baumbach, Binod B. Sahu, and Catherine Brooke

Soybean is one of the world’s most valuable crops and the U.S. is the world leader of soybean production. In 2010, the U.S. produced 35% of the world soybean crop followed by Brazil (27%) and Argentina (19%). The total U.S. soybean crop value was over $38.9 billion and U.S. exports of soybean and soya-products were over $23 billion in 2010. Soybean suffers yield suppression from various biotic stresses. In 2010, 14.4% of total yield valued $5.59 billion was suppressed by pathogenic diseases caused by microbes and nematodes. Soybean sudden death syndrome (SDS) is a major threat to soybean production in the U.S. Nationwide, the estimated soybean yield suppression from SDS in 2010 was 2.1% of total yield valued at $0.82 billion. The pathogen causing this disease, Fusarium virguliforme, stays in the soil and has never been identified from the above ground diseased foliar tissues. We have identified a proteinaceous toxin, FvTox1, which produces foliar SDS-like symptoms in soybean, only in presence of light. Expression of a single chain variable fragment (scFv) antibody against the toxin molecule in transgenic soybean plants enhanced the foliar SDS resistance. Recently, we have created F. virguliforme mutants lacking the FvTox1 gene by applying a homologous recombination approach. Analyses of five independent mutants through stem-cut and root inoculation assays revealed that FvTox1 is a virulence factor required for foliar SDS symptom development. FvTox1 accumulates in chloroplasts. FvTox1 binds to a soybean carbonic anhydrase isoform in yeast that transports carbon dioxide (CO2) in the form of carbonic acid into chloroplasts for photosynthesis. Presumably following binding of FvTox1 to carbonic anhydrase, the CO2 transport is impaired leading to formation of highly toxic free radicals which initiate foliar SDS symptoms development. Expression of the maize homologue of the protein lacking affinity to FvTox1 provides enhanced SDS resistance in transgenic soybean plants. Thus, most likely FvTox1 produces foliar SDS symptoms through interaction with a soybean carbonic anhydrase isoform. Applying Technology to Help Resolve Real-World Issues - The Case of Developing Low Phytate/High Available Phosphate Soybeans

Kristin Bilyeu, USDA-ARS, Plant Genetics Research Unit, Columbia, Missouri Vince Pantalone, Department of Plant Sciences, University of Tennessee, Knoxville, Tennessee

A rapidly evolving technology front presents many challenges to apply technology- driven advances to improve established complex development systems such as plant breeding efforts. Developing soybean lines with commercial feasibility in terms of agronomics and yield potential that also have one or more seed composition traits sets the stage for the development of systems that incorporate new knowledge about the molecular basis of those targeted plant traits. The molecular genetic basis has recently been determined for a number or seed compositional traits, and this knowledge has enabled the development of molecular marker technology to directly select for the alleles controlling the traits. Coupling this technology with emerging new information on fundamental agronomic traits such as maturity and plant architecture provides both forward and retrospective analyses of population development and selection strategies. As an example, progress has been made and will be shown for the development of soybean lines with the low phytate/high available phosphate trait. In addition, a view toward the future of soybean development aided by fundamental translational discoveries of gene function and the ability to rapidly incorporate new technology into plant breeding schemes will be presented. De-bugging Soybean

Wayne Parrott, University of Georgia María Ortega, University of Georgia John All, University of Georgia H. Roger Boerma, University of Georgia

Soybean is susceptible to a variety of leaf-chewing caterpillars and beetles, among other insects. An allele conferring resistance to leaf-chewing caterpillars and beetles via both antibiosis and antixenosis was detected as a QTL on the chromosome 7 of a Japanese soybean, PI229358. Map-based cloning and sequence comparisons to susceptible genotypes were used to identify the gene responsible for insect resistance. This gene has been called GmOruga, and is annotated as Glyma07g14530, a putative flavonoid 3-O Glycosyltransferase (UF3GT). The resistant allele codes for a truncated UF3GT protein, suggesting that specific flavonoids, or altered flavonoid profiles play a role in resistance to leaf-chewing insects. Besides conferring resistance by itself, GmOruga provides enhanced resistance when combined with either Bt, or the sharp- leaf trichome allele (Pb) found in PI227687, another insect resistant soybean from Japan. Also, when the resistant allele for GmOruga is present, two additional QTLs for resistance from PI229358 can be identified on chromosomes 12 (chr12) and 18 (chr18), which are antixenotic and antibiotic, respectively. Although GmOruga has not shown activity against other types of insects, the alleles on chr12 and chr18 each show field resistance against the kudzu bug (Megacopta cribraria), a recent invasive species in the South. Overall, effective resistance to various insects can be achieved by pyramiding different resistance genes. Discovering the mode of action of GmOruga may be useful for transferring insect resistance to other crops. Fine Mapping Rag1 and Rag2 and the Evaluation of New Aphid Resistance Sources

Brian Diers, University of Illinois Ki-Seung Kim, Tong Geon Lee, Jianping Wang, Carol Bonin, Curt Hill, Glen Hartman, and Matt Hudson

Soybean aphid (Aphis glycines Matsumura) was first discovered in North America in 2000 and has become an important pest of soybean [Glycine max (L.) Merr.]. After this discovery, sources of aphid resistance were identified from the soybean germplasm collection and the major resistance genes Rag1 and Rag2 were mapped. These genes were then fine mapped to develop markers closely linked to the genes for use in marker-assisted selection and to develop resources needed for gene cloning. Based on the Williams 82 genome sequence, Rag1 was mapped to a 115 kb interval that contained two genes predicted to encode nucleotide-binding site leucine-rich repeat (NBS LRR) proteins and Rag2 was mapped to a 54 kb interval containing one NBS LRR gene. The Rag1 interval was cloned and sequenced from Dowling, the source of the gene, revealing that this genotype has two NBS LRR genes and one of these genes was not in the Williams 82 interval sequence. Both candidate genes from Dowling were transformed into a susceptible genotype for functional validation; however, resistance has not been confirmed in plants transformed with either candidate gene. A second focus of our research was the identification of new aphid resistance genes from the soybean germplasm collection, a focus that has become more urgent since the discovery of soybean aphid biotypes that can overcome resistance genes. Populations segregating for aphid resistance genes from 23 plant introductions (PIs) have been evaluated. The resistance from 18 PIs was mapped to only the interval containing Rag2, resistance in three PIs was mapped only to Rag1, resistance was mapped to both Rag1 and Rag2 in one PI, and resistance was mapped to both the Rag1 and Rag3 intervals in one PI. We are currently investigating the use of markers tightly linked to these two resistance genes to predict if aphid resistant genotypes from the germplasm collection possess the known Rag1 or Rag2 resistance alleles. If successful, screening germplasm with these markers would allow us to focus resources on accessions that potentially have alternative alleles. To date, we have not identified any single marker or marker combinations that can be used to predict whether PIs have resistance at Rag2, showing that these predictions will require better marker resources. Transposition of The Maize Ds Element in the Soybean Genome

Tom Clemente, University of Nebraska-Lincoln Manmeet Singh, University of Nebraska-Lincoln Han Ngyuen, University of Nebraska-Lincoln Shirley Sato, University of Nebraska-Lincoln Truyen Quach, Fareha Razvi, Renee Schirmer, Yun-Tsi Bolon, Carroll, Vance

Soybean (Glycine max (L.) Merr.) is a major oilseed commodity which partitions carbon and nitrogen flux during embryogenesis towards two primary storage reserves, protein and oil, at approximately 40% and 20%, respectively in the seed. This attribute makes soybean a valuable feedstock in many food, feed and industrial applications. Over the past decade a wealth of genomic resources have been established for soybean that will aid in elucidating the underlying biology governing the growth and development of the crop. This in turn will foster innovative breeding and genetics approaches leading to improvements in agronomics and end-use quality. Loss- and gain-of function mutants are powerful resources that complement functional genomics programs. The maize two- component transposon system Ac/Ds has been used in many plant species as a means to generate insertional and activation tagged mutants. To assess the ability of the Ds- element to transpose in the soybean genome we produced approximately 550 F1 Ac- stacks with the Ds-activation tag element and 144 F1 Ac-stacks with Ds-enhancer trap element. Among 17 F2 derived populations from Ac X Ds-activation stacks we observed 26 unique germinal transpositions with an estimated 3.15% germinal transposition frequency. Whereas among 22 F2 derived populations from the Ac X Ds- enhancer trap stacks only six unique germinal transpositions were detected, translating to an estimated 0.5% transposition frequency. Based on sequence data collected from junctions about the transposed Ds elements it appears that in the soybean, Ds quite frequently re-inserts at unlinked positions respective to its corresponding launch site. Two germinal mutants characterized, a Ds-enhancer trap and a Ds-activation tag, landed in the third intron of a putative cyclic nucleotide binding domain gene, and a predicted IMP/GMP specific nucleotidase, wherein the former resulted in a reduction in tagged transcript accumulation, while the latter lead to miss-expression of the tag gene. Breeding the Epigenome in Soybean

Sally Mackenzie, University of Nebraska Sunil Kenchanmane Raju, University of Nebraska Ying-Zhi Xu, University of Nebraska Mon-Ray Shao, University of Nebraska

MSH1 is a plant-specific nuclear gene that encodes a product that functions in both mitochondria and chloroplasts. In the chloroplast, the protein appears to carry out two functions, one in DNA stability and the second in environmental sensing. RNAi suppression of MSH1 in sorghum and soybean produces a condition of developmental reprogramming that dramatically alters growth and phenotype in a manner that is heritable even in the subsequent absence of the RNAi transgene. Crossing these developmentally altered plants to their original wildtype form gives rise to progeny families that display a wide range of phenotypic variation in plant architecture, maturation, above-ground biomass, flowering and seed production traits. We have subjected this non-genetic variation to selection and found it to be highly heritable. We will present evidence to suggest that this process of epigenetic change is conditioned via chloroplast changes and may prove valuable agronomically. Dissecting Control of Carbon Partitioning in Developing Seeds by Enhancer Tagging: Characterization of Arabidopsis Mutants and Translational Work In Soybeans

Knut Meyer, DuPont Pioneer, Agricultural Biotechnology

During the maturation phase of seed development, photoassimilates, mainly sucrose and amino acids, are converted to the seed storage compounds starch, seed storage lipids, and proteins. A complete understanding of the properties of this pathway, specifically the control points influencing the partitioning of carbon between starch, oil, and protein, has been the focus of diverse research approaches due to the importance of seed storage compounds in human civilization as sources of food, feed, and fuel.

Our rapidly increasing understanding of the network of reactions that support anabolic pathways leading to oil, protein, and starch biosynthesis in developing seed is currently not reflected in the sophistication of transgenic approaches targeting the biotechnological modification of carbon partitioning in seed. For example, most transgenic approaches to increase the oil content of seed are composed either of attempts to increase ‘pull’ toward oil biosynthesis through the increased expression of genes controlling plastidic fatty acid biosynthesis or cytosolic glycerolipid assembly or by increasing metabolic ‘push’ in this direction through the overexpression of developmental or global metabolic regulators that enhance the expression of genes encoding enzymes of glycolysis and fatty acid biosynthesis.

I will describe some of our attempts to greatly increase the number of gene targets affecting carbon partitioning in developing seeds. Our goal was the identification of components of the seed filling machinery that divert carbon away from the accumulation of seed storage lipids, thus providing new gene targets for increasing the oil content of plant tissues through RNA interference. To this end, we screened enhancer tag libraries of Arabidopsis for dominant low-oil mutants. We used this approach because low oil content (increased seed density) is a phenotype amenable to low-cost, high-throughput screening using density gradients. I will describe the detailed characterization of mutants, the molecular identification of genes responsible for the low-oil phenotype and the utility of the identified gene targets to increase the oil content of plant tissues. Soybean Yield Potential and Genome Re-Sequencing: Stepping into the Final Frontier!

James Specht, University of Nebraska - Lincoln

At a 1981 CSSA Symposium on genetic contributions to crop yield gains, I reported that USA ON-FARM soybean yields had increased from 1924 to 1980 at a linear rate of 20.9 kg/ha per year. At a 1998 CSSA Symposium, 17 years later, the linear rate from 1924 to 1997 was slightly higher (22.3), and at a recent 2011 ASTA Conference, the linear rate from 1924 to 2010 was again higher (23.3). Recently, NC USA researchers conducted a multi-state, multi-site set of 2-year performance trials of 160 MG II, III, & IV cultivars that had been released by public/proprietary breeders during the past 80 years, and found that the GENETIC gain in yield improvement was about 20 kg/ha per year. Clearly, rapid producer adoption of new cultivars is a major driving force in the continuing rise of USA on-farm yields. Improvement in YIELD POTENTIAL requires the selection of genotypes with a steeper seed yield response to (abiotic-factor) resource availability differentiatings low- and high-yield production environments. Despite the vast amount of genomic data now available for soybean breeding, we still have no sound understanding of the molecular basis of how empirical selection for yield has resulted in ever-higher yield potential. Two USB-funded research projects are now underway that are expected to help us gain more understanding in that regard. The goal of one project is to determine if genomic "breeder signatures" indicative of intense to-date yield selection can be identified via a retrospective look at re-sequenced genomes of ancestral land races and historic milestone cultivars. The goal of other project is to determine if a nested association mapping (NAM) analysis of a genotypic and phenotypic data obtained on 40 NAM populations - each comprised of 140 F5-derived RILs - can be used to identify heretofore known favorable alleles at "yield potential" QTLs that are different from the alleles that have already been fixed in the current elite germplasm pool. In any event, we have reached the tipping point where greater amounts of genomic and genotypic data may not be gainfully useful, unless we begin to commensurately increase the amount of precisely measured phenotypic data by testing genotypes in multiple production environments. After all, the discipline of genetics is still based on the Mendelian "calibration" principle of "phenotypes are observed; genotypes are inferred", and despite the generation of vast amounts of genotypic data, sparse phenotypic data will impose limits on that calibration. Investigating Nitrogen Deficiency in Common Beans

Jamie O'Rourke, USDA-ARS, University of Minnesota Luis Iniguez, Universidad Nacional Autonoma de Mexico Bruna Bucciarelli, USDA-ARS, University of Minnesota Jenna Woody, Iowa State University Randy C. Shoemaker, USDA- ARS, Iowa State University; Michelle A. Graham, USDA- ARS, Iowa State University; Steven Cannon, USDA-ARS, Iowa State University; Phillip E. McClean, North Dakota State University; Scott A. Jackson, University of Georgia; Georgina Hernandez, Universidad Nacional Autonoma de Mexico; Carroll P. Vance, USDA-ARS, University of Minnesota

Phaseolus vulgaris (common bean) and soybean diverged from a common ancestor approximately 19 million years ago. The genome of P. vulgaris is approximately half the size of soybean, making it an excellent model for soybean genetics. Nitrogen (N) is often a growth-limiting nutrient and N deficiency results in reduced yield. N deficiency is also highly correlated with drought stress, an increasing problem due to global climate change. We performed next generation sequencing (RNAseq) on leaves, roots, and nodules of P. vulgaris cv. Negro jamapa plants that were inoculated with an effective rhizobium, an ineffective rhizobium mutant strain (fix-), or were provided nitrogen via fertilization. Pathways involved in N assimilation and utilization were examined to determine the effect of fertilization vs. symbiotic N fixation and N deficiency on gene expression patterns. We have identified sequences uniquely expressed in each tissue dependent on the source of available N. Additionally, we have identified the homologous sequences in soybean and have compared gene expression patterns between the two legume species. Transcriptome Analysis of Ozone-Response in Susceptible and Tolerant Soybean Lines

Jessica Schlueter, University of North Carolina at Charlotte Adam Whaley, University of North Carolina at Charlotte Kent Burkey, USDA-ARS Thomas Carter, USDA-ARS Jim Orf, UMN; Jaime Sheridan, UNC-Charlotte; Sajedeh Safari, UNC-Charlotte

As the global climate changes, plants will be challenged by environmental stresses that are more extreme and more frequent leading to increased yield loss. Specifically, ozone stress is an increasing problem in both urban and rural areas. Soybeans are one of the plant species that are quite ozone sensitive. The genetic mechanisms of response to ozone stress in soybeans are unclear and a good source of resistance in breeding stocks is lacking. We are working with two varieties of soybean, Fiskeby III (tolerant) and Mandarin (Ottawa, susceptible). We have collected and profiled transcripts (RNA- seq) from leaf tissues from both control and high ozone exposure at two time points. Illumina-based RNA-seq followed by a GO term enrichment was used to elucidate the differences in response mechanisms between varieties. Photosynthesis, stress response, and carbohydrate metabolism related GO terms are particularly enriched between the varieties at high ozone and within the same variety. The differences found in the GO enrichment analysis indicate that the differences in response between ozone tolerance and intolerance in Fiskeby is largely one of dosage of gene products and the timing of specific cellular responses to ozone exposure that limit or facilitate cell damage. DNA Replication and the Iron Deficiency Response in Soybean

Michelle Graham, USDA-ARS, CICGRU Sarah Atwood, Iowa State University Jamie O'Rourke, USDA-ARS, PSRU Gregory Peiffer, USDA-ARS, CICGRU Chunquan Zhang, Iowa State University; John Hill, Iowa State University; Steven Whitham, Iowa State University; Randy Shoemaker, USDA-ARS, CICGRU

Iron is a micronutrient required for proper growth and development. In soybean (Glycine max (L.) Merr.), iron deficiency results in interveinal chlorosis and decreased photosynthetic capacity, leading to stunting and yield loss. A recent microarray study identified Replication Protein A subunit 3 (GmRPA3c, Glyma20g24590) as one of the most differentially expressed genes in response to iron stress in leaves of two near- isogenic lines (NILs) differing only in their iron efficiency. In this study, gene expression analyses investigated the role of all soybean RPA subunits in the iron stress response. Nine RPA homologs were significantly differentially expressed in response to iron stress in the NILs Clark (iron efficient) and Isoclark (iron inefficient). Interestingly, the RPA homologs had opposing expression patterns in the two NILs, with RPA expression significantly repressed during iron deficiency in Clark. One RPA homolog, GmRPA3c, was positioned within an iron deficiency chlorosis (IDC) quantitative trait locus. Silencing of GmRPA3c/d in Isoclark, to mimic Clark expression, produced plants with improved IDC symptoms and chlorophyll content. We demonstrate that a suite of DNA replication genes, including the GmRPAs, is repressed by iron stress in the iron efficient line Clark. Further, we provide evidence that an introgression from the iron inefficient donor parent T203 elsewhere in the genome resulted in the misexpression of DNA replication genes in Isoclark. Our results suggest RPA proteins play a prominent role in iron stress response in soybean and support GmRPA3c as the candidate gene for the IDC quantitative trait locus on soybean chromosome 20. Exploitation of Root System Architecture for Improving Drought Tolerance

Henry T. Nguyen, National Center for Soybean Biotechnology and Division of Plant Sciences, University of Missouri, Columbia, Missouri, 65211

Drought is the major abiotic stress factor affecting yield and stability of crop production. It is reported that the average yield losses are more than 40% in soybean due to drought stress. Understanding the concept and components of drought resistance is a key factor for improving drought tolerance of crops. Being that soybean is one of the major food sources in the world, our long term goal is to produce drought tolerant soybean with improved productivity. Our research program focuses on an integrated genetics and genomics approach to dissect molecular processes from transcript to phenome. The root system plays a vital role in plant adaptation and productivity under water-limited environments. Deeper and proliferate root systems help extract enough water under these environmental conditions. We have screened and identified soybean lines which exhibit genetic diversity in root system developmental plasticity in response to water stress, in order to enable physiological and genetic analyses of the regulatory mechanisms involved. Comprehensive expression profiling using current “omic” technologies were performed to dissect molecular processes and genes associated with the root system architecture responses under water deficit conditions. Functional characterization of key candidates, engineering of selected genes through the translational genomics pipeline, and testing these transgenic plants to study associated physiological mechanisms are in progress. This presentation will highlight recent discoveries towards better root system architecture in soybean for enhanced stress resistance and yield. Making the Best Soybean Oil That We Can

John Browse, Washington State University Deirdre Fahy, Washington State University Shuangyi Bai, Washington State University James Wallis, Washington State University Chaofu Lu, Trisha Brock and Jennifer Watts, Washington State University

Soybean is one of many crops that accumulate seed reserves as oils composed of triacylglycerols. These vegetable oils constitute important sources of food and industrial products with commercial production worth $35 billion worldwide. To a large extent, the properties of different oils depend on the degrees of unsaturation of the fatty acid components and this is a function of desaturase enzymes that introduce double bonds into the fatty acid chains. There is considerable interest among plant breeding and biotechnology companies in producing modified soybean oils that address consumer and industry goals for high-oleic and low-saturate oils. Conventional and mutation breeding have provided some desirable alterations in the composition of several oilseeds. However, changes that require more sophisticated genetic engineering techniques require a detailed understanding of the biochemistry and regulation of oilseed triacylglycerol synthesis. Our work uses mutation and transgenic approaches in Arabidopsis to alter seed fatty acid composition and to investigate oilseed metabolism. We have cloned many different desaturases from Arabidopsis, from the nematode Caenorhabditis elegans, and from several microbes. I will summarize some of this work and describe how it contributes to developing strategies for altering seed oil composition. Our investigation of the rod1 mutant led to the discovery of phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) as a new enzyme of seed lipid metabolism. PDCT controls the availability of 18:1 for desaturation. Recent examples indicate that understanding and altering the enzymatic machinery of oilseeds will be a key to successful engineering of oil composition. Using A Novel Acyltransferase to Alter the Structure of Plant Triacylglycerols for Targeted Applications

Timothy Durrett, Kansas State University

The triacylglycerols (TAGs) found in the seed oil of most plants consist of three long chain fatty acids esterified to a glycerol backbone. However, some species synthesize unusual TAGs with acetate instead of a long chain fatty acid at the sn-3 position. The altered structure of these acetyl-triacylglycerols (acTAGs) confers them with different physical and chemical properties compared to regular TAGs, making them potentially useful for various industrial and nutritional applications. For example, the viscosity of acTAGs is lower than that of other seed oils, making them an ideal candidate for an enhanced straight vegetable oil (SVO) biofuel with improved combustion properties. From a nutritional standpoint, the absence of a third long acyl chain reduces the calorific content of these unusual TAGs. Thus acTAGs could be used as a reduced-calorie oil with a molecular structure similar to existing commercial products such as SALATRIM.

The seeds of the ornamental plant Euonymus alatus (Burning Bush) produce high levels of acTAGs. The enzyme necessary and sufficient for this synthesis was cloned using a transcriptional profiling approach and named EaDAcT (Euonymus alatus diacylglycerol acetyltransferase). Biochemical characterization of EaDAcT demonstrated that it uses acetyl-CoA, but not long chain acyl-CoAs, to acylate diacylglycerols. Expression of EaDAcT in Arabidopsis yielded transgenic lines with seed oil containing 45 mole% acTAGs. The fatty acid content of the transgenic seed was unchanged compared to wild type seeds. However, as the fatty acids in the transgenic seed were distributed among TAG molecules containing two as well as three fatty acids, the transgenic seed contained more TAG molecules per seed and were consequently larger and heavier. No germination defects were observed in these high acTAG lines. Additionally, analysis of lipid content demonstrated that acTAGs were efficiently utilized by the germinating seedlings. Expression of EaDAcT in different Arabidopsis mutants led to enhanced synthesis of acTAGs, with up to 65% of the seed oil comprised of acTAGs. Future work will focus on increasing these yields of acTAGs, as well as producing large quantities of acTAGs in oil seed crops for functional product testing. To this end, EaDAcT has been expressed in soybean and Camelina sativa. Analyzing Developing Soybean Seed Metabolism with Isotopic Labeling and Metabolic Flux Analysis

Doug Allen, USDA-ARS/Donald Danforth Plant Science Center

Isotopic labeling experiments provide biochemical descriptions of metabolism. In developing oilseeds, the use of isotopic labeling along with computational methods such as metabolic flux analysis can aid our understanding of carbon partitioning and clarify network operation. Soybeans produce significant amounts of both protein and oil from precursors generated through primary metabolism. Additionally, protein concentrations coincide with the carbon and nitrogen levels received by the developing embryo. We examined the relationship between the supplied carbon and nitrogen and seed composition through a series of embryo culturing experiments with unlabeled, 13C- or 14C labeled substrates. Three distinct ratios of carbon to nitrogen supply were further explored through metabolic flux analysis. The analyses indicate dramatic changes in protein level that will be discussed with respect to the results isotopic labeling and metabolic flux results. Development of High Oleic Soybean Oil

Anthony Kinney, DuPont Pioneer

Improving the fatty acid composition of soybean oils has been an active and highly successful effort on the part of many DuPont scientists beginning as far back as 1990. The showcase product from this work is Plenish High Oleic Soybean Oil, the first biotech product in soybean developed specifically for the benefit of consumers.

With about 75% oleic acid, the Plenish product was developed as a replacement for partially hydrogenated oils which are stable to oxidation but contain unhealthy trans fatty acids. Trans fat labeling requirements, which commenced in 2006, brought about a swift and dramatic change in the food industry as leading food companies and quick service restaurant chains attempted to convert their products out of partially hydrogenated oils to meet the labeling deadline. While satisfying the trans-free labeling requirement, many food manufacturers are now dissatisfied with their current oil selections. Experts expect another wave of conversions in the next few years as first generation trans solutions are replaced by more stable products like Plenish. This time, the major driver in the conversion will be cost reduction.

The very same properties that make the oil an attractive option for the food industry, also provide benefit in the industrial markets. For example, for use in both lubricants and transformer fluids, the oxidative stability of the Plenish product provides utility in applications which require stability beyond that of conventional plant oils. Market studies predict very strong growth of high oleic oils in these applications as companies begin to replace petroleum-based products with sustainable, environmentally-friendly solutions. Beyond stability, the homogenity of the oleic-based oils have benefit for adhesive, polyol and other oleochemical products. Functional Characterization of Soybean Transcription Factors Using Comparative and Genomic Approaches

Marc Libault, University of Oklahoma Andrew Farmer, National Center for Genome Ressources Trupti Joshi, University of Missouri Laurent Brechenmacher, University of Missouri Dong Xu, University of Missouri; Gregory May, National Center for Genome Resources; Gary Stacey, University of Missouri

Transcription factors (TFs) regulate the expression of a large number of genes and control plant development and plant response to environmental stimuli. Hence, they are essential actors in transcriptional regulatory networks. In soybean, over 5600 TF genes were identified based on conserved TF signature domains. Taking advantage of three different resources: 1- the soybean genome sequence; 2- soybean transcriptomic databases; 3- knowledge gained in other models (e.g. Arabidopsis thaliana, Medicago truncatula and Lotus japonicus); we used a comparative genomic approach coupled with transcriptomic data sets to identify soybean transcription factor genes controlling soybean shoot development, soybean flower development and soybean nodulation. This powerful approach supports to the likelihood of common ancestral function of transcription factor genes between plant models by highlighting their conserved expression patterns.

The characterization of the function of transcription factor genes is also challenging due to their low level of expression and their cell-type-specific expression profile. Hence, a whole tissue which is a combination of cells with different fates and different expression profiles is not an adequate plant material to map transcription factors clearly into regulatory networks. To resolve this problem, soybean root hair cells have been used as a single cell type model to study soybean root cell infection by Bradyrhizobium japonicum, the symbiotic bacteria involved in soybean nodulation. This approach allowed us to better characterize TF genes controlling the early events of soybean nodulation including new differentially regulated transcription factor genes as well as orthologs of NSPs, NINs and HAP2-1, M. truncatula transcription factors well- characterized for their role in nodulation. max Mutation: mPing-based Gene Discovery

C Nathan Hancock, University of South Carolina Aiken Wayne Parrott, Melinda Yerka, Christie Kemp, Peter Lafayette, Kristen Floyd, and Donna Tucker (University of Georgia); Tom Clemente (University of Nebraska-Lincoln)

Development of genomics tools that connect traits to specific genes will result in benefits for all plant breeders and researchers. To this end, a relatively new mutagenesis system based on the mPing transposon from rice is being deployed in Glycine max. The mPing element is a relatively small (430bp) DNA transposon and is mobilized by the transposase proteins encoded by the Ping or closely related Pong elements. Transforming mPing and the Ping transposase genes into soybean produced lines that exhibit active mPing transposition. Characterization of the heritable mPing insertions produced by these lines indicated that mPing transposed to unlinked sites and preferentially inserted into euchromatin, with over 70% landing in or near annotated genes. Large numbers of these plants are being screened for mutant phenotypes and bulked seeds from these lines are available for distribution. High-throughput methods for insertion site determination are also being developed to facilitate analysis of mPing mutagenized plants.

Now that that the potential of the mPing-based tagging system has been established, the focus is to increase its effectiveness. In order to increase the overall transposition rate, new lines expressing modified Pong transposase genes that show higher transposition frequency in a yeast transposition assay are being produced and screened. Tissue culture treatment is also being tested as a method to increase the transposition rate. In addition to these strategies, mPing-based activation tags designed to induce overexpression of nearby genes are being developed. A number of activation tagging constructs have been made and tested in yeast to determine how modification of mPing affects its mobility. These constructs are currently being tested in soybean and should significantly improve the mutation rate. How Resilient Is the Soybean Genome? Insights from Fast Neutron Mutagenesis

Yung-Tsi Bolon, University of Minnesota Adrian Stec, Jeffrey Roessler, Jean-Michel Michno, Justin Anderson, Gary Muehlbauer, Carroll Vance, Robert Stupar

Previously, we described the development of a fast neutron mutant population resource in soybean and identified mutations of interest through phenotypic screening. Here, we consider the resiliency of the soybean genome by examining genomic rearrangements and mutations that arise from fast neutron radiation damage and repair. We previously documented deletions and duplications in a limited number of lines by comparative genomic hybridization and exome resequencing. Approximately two copy number variation events that ranged from one kilobase to three megabases in size were discovered per mutant line. Next-generation sequencing data on the genomic DNA of select mutant lines and data on over a hundred comparative genomic hybridizations has now been generated. Parallel studies were performed on a subset of mutant lines that showed no visible phenotype. Analyses of these datasets enable characterization of translocations, inversions, insertions, and point mutations in addition to deletions and duplications sustained in the soybean genome. These studies locate fragile or adjustable sites on soybean chromosomes, provide insight on gene essentiality and function, and contribute to the demarcation of a minimum soybean genome. Discovery of Gene Networks Regulating Soybean Defenses Using Virus-Induced Gene Silencing

Steven Whitham, Iowa State University Ajay Pandey, NABI Jianzhong Liu, Zhejian Normal University Chuquan Zhang, Alcorn State University Chunling Yang, Iowa State University; John H. Hill, Iowa State University; Michelle A. Graham, USDA-ARS; Kerry F. Pedley, USDA-ARS

Soybean production is dramatically affected by a variety of pathogens, including viruses, bacteria, fungi, oomycetes, and nematodes. These pathogens elicit a variety of responses in soybean cells that lead to susceptibility or resistance depending on the genotypes of the soybean cultivar and the pathogen isolate. The responses are controlled by complex networks of genes that mediate compatible and incompatible responses that determine the outcome of each interaction. While plant-pathogen signaling networks are being worked out in model plants, such as Arabidopsis thaliana, knowledge has lagged behind in crop plants, such as soybean. The soybean genome sequence, accumulation of various omics data sets on soybean-pathogen interactions, and the development of robust functional genomics tools, such as virus-induced gene silencing, provide the opportunity to rapidly accelerate our understanding of soybean gene networks that mediate interactions with pathogens. These resources coupled with information from model systems have enabled us to develop frameworks for gene networks that mediate resistance to soybean rust and Soybean mosaic virus. Small RNAs as Regulators of Nodulation

Janine Sherrier, University of Delaware

Small noncoding RNAs are potent regulators of plant gene expression, and their sequence homology with targets provides precise control of gene expression. We have identified small RNAs which target defense related genes and will present a model of how small RNAs may facilitate the interaction with beneficial microbes such as rhizobia. Soybean Root Hairs: A Single Cell Model for Plant Systems Biology

Gary Stacey, University of Missouri

We are continuing our characterization of soybean root hairs, with a specific focus on understanding the mechanisms by which the symbiont, Bradyrhizobium japonicum, infects the root to establish a nitrogen fixing symbiosis. Root hairs are single cell extensions of the root epidermis, which function primarily to increase root surface area and enhance water and nutrient uptake. However, they are also the preferred site for rhizobial infection. Previous research has characterized the root hair transcriptome, metabolome, and proteome in response to B. japonicum infection. These data are available publicly via soykb.org. Functional analysis has shown that silencing several genes identified in these previous studies results in severe defects in soybean nodulation. More recent work has focused on characterizing the root hair phosphoproteome, as well as the role of small RNA in controlling key rhizobial infection processes. In the case of the phosphoproteome, global proteomic analysis identified a total of 1126 phosphoproteins in root hairs of which 240 were found to respond significantly to rhizobial infection. We are now pursuing methods to identify the specific protein kinases responsible for these phosphorylation events. High throughput sequencing of small RNA libraries derived from root hairs, as well as the associated roots, identified a total of 178 miRNA species, including 47 novel miRNAs, including a few that appeared to be exclusively expressed in root hairs. The expression levels of 78 miRNAs were shown to respond to rhizobial infection. We also sequenced a degradome library and identified 459 mRNA targets for the various miRNAs identified. Ectopic expression of selected miRNA or silencing of the predicted target mRNAs resulted in significant effects on nodulation, suggesting a critical role for miRNA in regulating the nodulation process. In addition to these and other ongoing projects focused on the soybean -rhizobial symbiosis, we are also using our soybean root hair system to investigate the plant cellular response to abiotic stress. The ultimate goal of this research is a systems-level understanding of the soybean root hair, a single cell model. Glycome Profiling Of Soybean Root And Root Hair Cell Walls

Russell Carlson, Complex Carbohydrate Res. Center, Univ. of Georgia, Athens, GA Artur Muszynski, Complex Carbohydrate Res. Center, University of Georgia Sivakumar Pattathil, Complex Carbohydrate Res. Center, University of Georgia Utku Avci, Complex Carbohydrate Res. Center, University of Georgia

Malcolm O'Neill, Complex Carbohydrate Res. Center, University of Georgia; Marc Lebault, National Center for Soybean Biotechnology, University of Missouri; Laurent Brechenmacher, National Center for Soybean Biotechnology, University of Missouri; Will York, Complex Carbohydrate Res. Center, University of Georgia; Michael G. Hahn, Complex Carbohydrate Res. Center, University of Georgia; Gary Stacey, National Center for Soybean Biotechnology, University of Missouri

Soybean root hairs and roots stripped of root hairs were isolated from plants inoculated with Bradyrhizobium japonicum and from uninoculated plants. The hairs from inoculated (HIN) and uninoculated (HUN) soybean, and the stripped roots from inoculated (RIN) and uninoculated (RUN) soybean) were then ground to a powder in liquid nitrogen. These powders were sequentially treated with (i) potassium phosphate, pH 7, (ii) endopolygalacturonase (EPG) and pectin methyl esterase (PME), (iii) a xyloglucan-specific endoglucanase (XEG), (iv) 2 M imidazole-HCl, pH 7, (v) 1 M KOH, and (vi) 4 M KOH. The total carbohydrate content of the material solubilized by these six treatments and the final insoluble residue from HIN, HUN, RIN, and RUN were determined. A standardized amount of carbohydrate from each fraction was also used for glycome profiling using a panel of 150 monoclonal antibodies (mAbs) that recognize plant cell wall epitopes. Additionally, the glycosyl residue compositions of each of the seven fractions from HIN, HUN, RIN, and RUN were determined. Theses analyses (extraction, mAb-binding, and composition analysis) were repeated four times and the data evaluated statistically. Comparison of the HUN/RUN fractions showed significant increases in binding of mAbs that recognize xyloglucans. Increases were also noted in three mAbs that recognize galactomannan. A mAb that recognizes xylan showed a decreased binding. Fewer and less intense differences in mAb-binding were observed with HIN and HUN fractions. However, matrix-assisted time of flight mass spectrometry (MALDI-TOF MS) of selected HUN and RUN fractions showed differences in xyloglucan oligosaccharide subunit compositions. Those mAbs that showed discernible differences in binding to HUN/RUN and HIN/HUN fractions during glycome profiling are now being used for immunocytochemical analyses of soybean root sections generated from inoculated and uninoculated plants. These data will be described with regard to possible structural differences between root hair and root cell walls and changes in cell walls during the infection process. (Supported in part by DOE grant DE-FG02- 09ER20097 to the Complex Carbohydrate Research Center.) Basic and Applied Understanding of Signal Molecules from Rhizobia (Lipo- chitooligosaccharides) Contributes to Better Crop Production

Stewart Smith, Novozymes BioAg Bret Gygi, Novozymes BioAg Ahsan Habib, Novozymes BioAg Yaowei Kang, Novozymes BioAg

LCOs (Lipo-chitooligosaccharides, a.k.a. Nod factors) are signal molecules produced by rhizobia that are essential for the nodulation process in legumes. The resulting nodulation on soybeans provides symbiotic nitrogen fixation for plant growth and yield. Formulations for soybeans combining the LCO and rhizobia (Bradyrhizobium japonicum) have been developed and commercialized. LCO laboratory bioactivity is examined by both root hair deformation of Siratro (Macroptillium atropurpureum) and Gus reaction with Lotus japonicus LjCbp1-gus. LCO seed application on non-legumes, including corn, cotton, and wheat, also demonstrate increased growth parameters to include root and shoot dry biomass, and root length, surface area, and volume in greenhouse studies, and enhanced yields in field trials. Foliar applications of LCOs on legume (soybean) and non-legume (corn) in greenhouse and field trials also improved plant growth parameters. LCO applications with legumes and non-legumes applied as treatments to seed, in-furrow, and foliar spray have provided improved plant growth and yield responses. Genetic Control of Symbiosis Specificity in Soybean and Medicago

Hongyan Zhu, Department of Plant and Soil Sciences, University of Kentucky

Legume plants are able to engage in root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This mutualistic association is highly specific, such that each rhizobial species/strain interacts with only a specific group of legumes, and vice versa. Symbiosis specificity can occur at multiple phases of the interaction, ranging from initial bacterial attachment and infection to late nodule development associated with nitrogen fixation. Genetic control of symbiosis specificity is complex, involving fine- tuned signal communication between the symbiotic partners. We will present our recent work on positional cloning of Rj2, Rj4, and Rfg1 genes in soybeans that control nodulation specificity with different rhizobial strains. We will also report our progress in cloning genes in Medicago that regulate symbiosis specificity at the nitrogen-fixing phase. Genome Divergence in Glycine max and Glycine soja

Suk-Ha Lee, Seoul National University

From hundreds of different edible crops around the world, soybean (Glycine max) has been playing an important crop because of its versatility. There are many soybean varieties with different phenotypes that may have been influenced by evolutionary events and gave rise to genetic diversity from the ancestors. Studies on domestication history in the relation to population divergence have yielded the molecular and genetic evidence for the origin of species. Based on the complete sequence of G. max and G. soja, the undomesticated ancestor of G. max (in particular, G. soja var. IT182932), we were able to perform an in-depth study of the time divergence in a total of 12 soybean genotypes (6 G. max and 6 G. soja) and in 4 soja genotypes by applying next generation low coverage and deep sequencing technologies, respectively, which were selected after the construction of the genetic diversity analysis of SSR dendrogram and neighbor joining tree of choloroplast DNA SNPs using 104 wild and landrace soybeans, from China, Korea and Japan. Data from the results of both sequencings were used for evaluation of sequence variation and haplotype diversity among soybean populations. The additional 4 G. soja genotypes with the maternal inheritance of cp DNA selection criterion were analyzed for further investigation of genetic differentiation in the undomesticated ancestor of G. max. Single nucleotide polymorphisms identified as nonsynonymous change in protein coding regions from each genotype were used for calculating an approximate divergence time to look at the complexity of G. max and G. soja divergence, which can be very useful for the investigation of evolutionary events based on the molecular clock technique. Status of the Soybean HAPMAP and the Structure of High-Resolution Haplotype Blocks in the Soybean Genome

Qijian Song, Soybean Genomics and Improvement Laboratory, USDA, ARS, Beltsville, MD 20705 David Hyten, Pioneer Hi-Bred International Inc, 7000 NW 62nd Ave. Johnston, IA, 50131 Gaofeng Jia, Soybean Genomics and Improvement Laboratory, USDA, ARS, Beltsville, MD 20705 Randall Nelson, USDA-ARS, Soybean/Maize Germplasm, Pathology and Genetics Research Unit and Department of Crop Sciences, University of Illinois, Urbana, IL 61801 Charles Quigley, USDA-ARS, Soybean Genomics and Improvement Lab, Beltsville, MD; Edward Fickus, USDA-ARS, Soybean Genomics and Improvement Lab, Beltsville, MD; Perry Cregan, USDA-ARS, Soybean Genomics and Improvement Lab, Beltsville, MD

A haplotype-based map constructed based upon the analysis of an extremely large population offers a powerful approach for mapping of important traits via association analysis of traits with the mutations and ancestral haplotypes from which they arose. Using a high-density Illumina Infinium iSelect Beadchip, SOYSNP50K, with an average distance between adjacent SNPs of 9.1kb and 49.1kb in the euchromatic and heterochromatic regions, respectively, we genotyped 19,657 soybean accessions from the USDA Soybean Germplasm Collection maintained at Urbana, Illinois. The genotypes include 18,489 G. max accessions (Asian landraces, cultivars and other germplasm) and 1,168 G. soja accessions. A total of 42,509 SNPs were polymorphic with MAF>0.01 in G. soja or G. max and with an overall missing and ambiguous call rate of less than 1.4%. Among these, 77% of the SNPs have a minor allele frequency >10%. A total of 362 (30%) and 4305 (23%) of the accessions are redundant or highly similar (>99.9%) among the G. soja and G. max collections, respectively. The data are being used to compare linkage disequilibrium, haplotype block size and frequency among wild soybean, landraces and elite cultivars, to study the association of haplotype blocks with domestication, recombination hotspots and important traits and to select tag-SNPs and a core set of germplasm accessions. Genome Structure Variation in Glycine species

Jungmin Ha, Purdue University

The genus Glycine includes Glycine max, soybean, which is one of the most important crops for its nitrogen fixation capacity through symbiosis with soil-borne microorganisms. However, the narrow genetic diversity of elite cultivars poses a potential threat to disease. Therefore, the development of genomic tools for wild Glycine species has been undertaken so that soybean research community can have the infrastructure to find useful genetic variation and move it into cultivated soybean. Sequence information is limited in what it can tell us about genome structural variation due to the limitation of current sequencing technology. Thus, a FingerPrinted Contig (FPC) physical map was constructed and integrated with the draft sequence of G. max as a framework for genomic research. The FPC map covers up to 95% of the soybean draft genome sequence (gmax1.01) incorporating 4,628 genetic markers to align the physical maps with genetic linkage maps. The set of minimum number of BAC clones covering the most of the draft sequence was selected. In addition, a FPC map of Glycine soja was constructed and aligned to G. max draft sequence map using BES information. To detect chromosomal variation between G. max and G. soja, the FPC contigs of G. soja aligned to 2 or more chromosomes of G. max were chosen as candidate contigs covering potential chromosomal rearrangements. Computationally defined chromosomal rearrangements, such as reciprocal translocations, are being confirmed by Fluorescent in situ Hybridization (FISH). As more than 50% of soybean genome consists of repetitive sequences, which can confound FISH signal detection, primers will be designed around the candidate breakpoints targeting chromosome specific sequences as the FISH probes. Finally, tandem repetitive sequences located at centromeric regions will be identified in wild Glycine species already sequenced, including Glycine canescens, Glycine cyrtoloba, Glycine falcata, Glycine stenophita, Glycine syndetika, and Glycine tomentella. As centromeric repeats consist of mega- base array of tandem repeats in most higher plants, these sequences cannot be assembled with current sequencing technology. Tandem Repeat Finder data from the BESs and k-mer analysis data from the NGS were combined and one centromeric repeats in G. falcata was identified. This study will shed light on chromosome structural variation in the Glycine species and provide a framework for comparative genomics, gene cloning and evolutionary analyses of legume genomes. Evolution of A Complex Disease Resistance Gene Cluster in Soybeans and Relatives

Ashley Egan, East Carolina University Tom Ashfield, Bernard Pfeil, Roger Innes

Disease resistance is an important mechanism for plant survival and is conferred via disease resistance (R) gene families, which often proliferate and evolve both within and between species. In plants, whole genome duplication (WGD), or polyploidy, may disproportionately influence the evolution of R-genes. We used a comparative genomics approach to investigate the impact of polyploidy on and evolution of a complex nucleotide-binding (NB)-leucine-rich repeat (LRR) gene cluster that is associated with several disease resistance genes of known function, including Rpg1b (for Resistance to Pseudomonas glycinea1b), an R gene effective against specific races of bacterial blight, found in soybean (Glycine max) and common bean (Phaseolus vulgaris). Soybean has undo gone at least two rounds of whole genome duplication, one just prior to the diversification of the genus about 10-12 mya and one shared with common bean that took place about 50-60 mya. Our analyses showed that the three functional R-gene domains, the amino-terminal coiled-coil (CC) domain, the central nucleotide-binding domain (NB-ARC [for APAF1, Resistance genes, and CED4]), and the carboxyl-terminal LRR domain, have evolved along distinct evolutionary trajectories. We examined the extent of recombination or sequence exchange within and among R-genes using a series of recombination detection methods. Our results suggest that sequence exchanges within the NB-ARC domain were rare but interparalogue exchanges involving the CC and LRR domains were common, suggesting that these regions may be coevolving with pathogens. Analyses of positive selection found evidence thereof within each of the three domains, but an overrepresentation of positively selected residues was found in the predicted solvent-exposed face of the LRR domain. Mapping of positively selected sites onto predicated tertiary structure determined that most are located on the surface, suggesting a role in interactions with other proteins or domains. In reference to the impact of WGD, NB-LRR genes have been differentially lost or partitioned among Glycine homoeologues following the 10-12 mya WGD event. The single orthologous region in common bean contains about the same number of paralogues as found in the two soybean homoeologues combined. We conclude that while WGD in Glycine has not driven a stable increase in family size for NB-LRR genes, it has generated two recombinationally isolated clusters, one of which appears to be in the process of decay. Population Resequencing Reveals Evolutionary Propensities of the Soybean Genomes

Jianxin Ma, Department of Agronomy, Purdue University Jianchang Du, Department of Agronomy, Purdue University Zhixi Tian, Department of Agronomy, Purdue University Meixia Zhao, Department of Agronomy, Purdue University Yi Sui, Department of Agronomy Purdue University; Qijian Song, USDA, ARS, BARC- West; Steven Cannon, USDA ARS Corn Insect and Crop Genetics Research Unit; Perry Cregan, USDA, ARS, BARC-West

The paleopolyploid soybean has several striking genomic features, such as extensive genome reshuffling after the recent whole genome duplication (WGD) event and extremely contrasting distribution patterns of transposable elements (TEs) and genes between pericentromeric regions and chromosomal arms. However, the evolutionary forces that govern the divergence, retention, and distribution of duplicated genes in these two distinction chromatin environments and the biased accumulation of TEs in the pericentromeric regions of the soybean genome are poorly understood. We have recently examined the evolutionary rates and expression levels of duplicated genes vs. singletons using resequencing data from 31 wild and cultivated soybean accessions and compared the distribution patterns of TEs accumulated in the reference genome and the nonreference TEs in the 31 resequenced soybean accessions. We found that the genes in pericentromeric regions the cold spots for meiotic recombination showed significantly lower rates of non-synonymous substitution and higher levels of expression than their homoeologs in chromosomal arms. This asymmetric evolution of two members of individual WGD-derived gene pairs, echoing the biased accumulation of singletons in pericentromeric regions and TEs, suggests that distinct genomic features between the two distinct chromatin types are important determinants shaping the patterns of divergence and retention of WGD-derived genes and the distribution of TEs in this complex palaeopolyploid species. Exploring Structural Variation in the Soybean Genome

Robert Stupar, University of Minnesota Leah McHale, Ohio State University Carroll Vance, USDA-ARS and the University of Minnesota

Genome-wide structural variation, such as large deletions and duplications, are hypothesized to drive important phenotypic variation. Recent advances involving array hybridization and targeted resequencing now allow for the genome-wide assessment of structural and gene content variation within a species. We have used these methods to assess natural and induced genome variation in soybean cultivars and mutant lines. High rates of natural structural variation have been observed within gene-rich regions that harbor clustered multi-gene families, particularly the Nucleotide Binding (NB) and Receptor-Like Protein (RLP) classes, both of which are important for plant biotic defense responses. The co-localization of structural variants with elements of the plant defense response signal transduction pathways provides insight into the mechanisms and frequencies of soybean resistance gene evolution. Emerging technologies may soon allow for the establishment of genetic stocks with targeted structural variants that can address new hypotheses about the relationship between structural variation and disease gene evolution in soybean.

QTL Controlling Aluminum Tolerance in Soybean: SNP Marker Discovery and Validation

Hussein Abdel-Haleem, Institute of Plant Breeding, Genetics & Genomics. University of Georgia H. Roger Boerma, Institute of Plant Breeding, Genetics & Genomics. University of Georgia Thomas E. Carter, USDA-ARS, Department of Crop Science, North Carolina State University Tom Rufty, Department of Crop Science, North Carolina State University Zenglu Li, Institute of Plant Breeding, Genetics & Genomics, University of Georgia

Aluminum (Al) toxicity is one of the abiotic stresses that affect soybean production in the acidic soil zones throughout the world. Neutralizing soil pH by adding lime is impractical due to the high practice costs, the limiting effect on topsoil only, and the risk of environmental pollution. Selection of tolerant genotypes in an applied breeding program requires understanding the inheritance of Al tolerance and developing robust markers at QTL for MAS. A pervious report identified QTL for Al tolerance with the favorable alleles inherited from PI 416937 using RFLP markers in a population of Young x PI 416937. The population was genotyped with 164 SSRs to enhance the power of QTL detection and identify robust markers for MAS. With SSR markers, three QTL were detected that explained 59% of the phenotypic variation in tap root growth ratio under Al stress in the hydroponic system. A major QTL, qAL_PC_08, controlling 41% of phenotypic variation in tap root length ratio under Al stress was identified at chromosome Gm08 with the positive allele from PI 416937 for Al tolerance in the hydroponic system. A candidate gene approach was used to develop functional markers associated with Al tolerance for MAS. Six homologues for citrate synthase genes (CS) were found in soybean genome sequence at Gm02, Gm08, Gm14, Gm15, and Gm18. The CS homologue on Gm08 was sequenced in Young and PI 416937 and SNPs were detected. A simple probe assay (melting curving method) for Glyma08g42400-SNP (GSM0002) was developed that was co-localized with the major QTL on Gm08 for the Al tolerance trait. A set of RILs derived from Benning x PI 416937 was used to validate the results with GSM0002 and confirmed the association between this SNP and Al tolerance. The SNP and other SSR markers identified from this region on Gm08 could be used for MAS of Al tolerance.

Poster Number: 1 Analysis of the Xylem Sap Proteomes for Identifying Candidate Fusarium virguliforme Toxins Involved in Sudden Death Syndrome Development in Soybean

Nilwala Abeysekara, Department of Agronomy, Iowa State University, Ames, Iowa 50011 Madan Bhattacharyya, Department of Agronomy, Iowa State University, Ames, Iowa 50011

Sudden death syndrome (SDS) caused by the ascomycete fungus, Fusarium virguliforme, is one of the top four yield reducing diseases in soybean. The pathogen causes both root necrosis and foliar SDS. However, the pathogen has never been detected in the diseased foliar tissues. Foliar SDS is believed to be caused by host selective toxins, including FvTox1, secreted by the fungus. We investigated if the xylem sap of F. virguliforme-infected soybean seedlings contains secreted F. virguliforme- peptides, which could be involved in foliar SDS development. We applied LC-MS/MS in analyzing the proteomes of the xylem saps collected from either healthy or F. virguliforme-infected soybean seedlings and identified seven F. virguliforme peptides with secretion signal peptides. The same seven peptides were also identified from the F. virguliforme culture filtrate that causes foliar SDS, when fed to cut soybean seedlings. One of these peptides showed high sequence identity to cereto-platanin, a phytotoxin produced by Ceratocystis fimbriata f. sp. platani that causes canker stain disease in the plane tree. Of the 135 soybean proteins identified, 14 were found only in the xylem sap of the F. virguliforme-infected soybean seedlings and 16 were in the sap of healthy seedlings. This study suggested that multiple F. virguliforme proteins could be involved in foliar SDS development. Furthermore, a number of soybean sap proteins could be differentially accumulated in the infected soybean seedlings in response to F. virguliforme infection. This proteomic study revealed several peptides whose roles in the soybean-F. virguliforme interaction could be crucial in developing foliar SDS.

Poster Number: 2 Mapping Quantitative Trait Loci Encoding Partial Resistance To Phytophthora sojae In Soybean

Nilwala Abeysekara, Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa Rashelle Matthiesen, Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa Silvia Cianzio, Department of Agronomy, Iowa State University, Ames, Iowa 50011 Madan Bhattacharyya, Department of Agronomy, Iowa State University, Ames, Iowa 50011 Alison E. Robertson, Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa

Stem and root rot caused by the oomycete pathogen Phytophthora sojae, is an economically important disease of soybean across the world. The disease is managed primarily by planting cultivars with single-gene mediated resistance. However, this type of resistance is becoming ineffective due to the emergence of new pathogen races. Partial resistance (PR) or field tolerance to P. sojae, which is polygenic in nature, has also been reported in soybean. It provides broad-spectrum, low level of root resistance against all physiological races of the pathogen. Therefore, incorporation of PR into soybean cultivars would provide a more durable form of disease management. The main objectives of this study were to identify molecular markers linked to quantitative trait loci (QTL) for PR to P. sojae (PRPS), and to validate the rice method as a more objective screening method for PR. Two recombinant inbred line populations were developed by crossing the plant introduction, PI399036, with two germplasm lines, AR2 and AR3. PI399036 carries high level of PRPS. AR2 and AR3 show low PRPS but carry genes for resistance to iron deficiency chlorosis. Both populations were advanced to the F7 generation and screened for PRPS using the rice method. Roots of both the P. sojae-infected and uninfected plants were evaluated 30 days after planting using a WinRhizo root scanner. Dry root and shoot weights were also obtained to quantify the levels of PR in the populations. Data analysis is in progress.

Poster Number: 3 Expression of Cry1Ac Gene Driven by Arabidopsis Rubisco Small Subunit 1A Promoter Conferred Resistance to Helicoverpa armigera in Chickpea (Cicer arietinum)

Sumita Acharjee, Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat 785013 Assam India Bidyut Kumar Sarmah, Department of Agricultural Biotechnology, Assam Agricultural University, Jorhat 13 P. Ananda Kumar, National Research Centre for Plant Biotechnology, Indian Agricultural Research Institute, New Delhi 12, India Joel Armstrong, CSIRO Entomology, GPO BOX 1700, Canberra, ACT 2601, Australia Willian J. Moar, Monsanto Company, 800 North Lindberg Blvd, St. Louis, MO 63167, USA; Andy Moore, T J V Higgins, 5CSIRO Plant Industry, GPO Box 1600, Canberra, ACT 2601 Australia

Transgenic chickpea (Cicer arietinum) plants were produced using a chimeric Cry1Ac gene to confer resistance to pod borers (Helicoverpa armigera). Cotyledonary explants containing half embryonic axes were infected with Agrobacterium tumefaciens harboring a binary vector. The vector contains an nptII gene as the selectable marker and a chimeric insecticidal Cry1Ac gene driven by the Arabidopsis rubisco small subunit (SSU) gene (ats1A) promoter. After co-cultivation with Agrobacterium, the explants were transferred to regeneration and selection medium. The nptII gene allowed in vitro selection of putative transformed shoots on a high dose (200mg/L) of kanamycin. We generated 29 independent transgenic lines cotransformed with both nptII and Cry1Ac genes. These lines expressed Cry1Ac protein at varying levels in the leaves and were grouped as low, medium and high expressors. Of the 29 transgenic lines 12 lines transmitted the Cry1Ac transgene into the next generation at a 3:1 ratio indicating a single copy of transgene insertion. Phenotypic data including seed yield per plant showed near normal phenotypes of lines expressing high levels of Cry1Ac protein. The high expressing lines, BS81G, BS100B, and BS100E, showed complete resistance to pod borer larvae in insect bioassays. These lines are now being back-crossed to transfer the transgene into different genetic backgrounds.

Poster Number: 4 Towards Understanding Gene Regulatory Networks Underlying Seed Maturation and Storage Reserve Production

Yong-Qiang Charles An , Plant Genetics Research, ARS-USDA at Donald Danforth Plant Science Center, St. Louis, MO 63132 Wolfgang Goettel, Plant Genetics Research, ARS-USDA at Donald Danforth Plant Science Center, St. Louis, MO 63132 Zhenhai Zhang, Plant Genetics Research, ARS-USDA at Donald Danforth Plant Science Center, St. Louis, MO 63132 Ronglin Wang, US EPA, Cincinnati, OH

Seed maturation and its accompanying seed reserve production are accomplished through the concerted activity of many gene products and biological pathways. However, their underlying transcriptional gene regulatory networks remain unknown. We examined polyadenylated messenger RNAs, small RNAs, and phytohormone and lipid profiles of cotyledons at six distinct seed maturation stages, and inferred 305 transcription factor regulatory networks (modules) through integrating them with additional 115 transcriptomes of various soybean seed related tissues, and regulatory cis-elements in each module. Further analysis of small RNA sequence reads identified 129 novel cotyledon microRNAs, and revealed that 54 miRNAs were differentially regulated over the course of seed maturation. A rich presence of intronic miRNAs and a high miRNA gain/loss rate were observed in cotyledons, suggesting that introns are preferential locations for new miRNAs to arise. Expression patterns of the cotyledon miRNAs and their predicted target transcripts were compared to predict their functional interactions in cotyledons based on the negative correlation of their expression patterns. The topologies of the cotyledon miRNAs in the transcription factor gene networks and the biological pathways that are potentially regulated by each transcription factor network were determined. In addition, we have compared transcriptomes of soybean and Arabidopsis seeds, and provided an insight into the conservation and divergence of their underlying transcriptional regulatory programs. The knowledge of the gene regulatory networks and identification of the key regulatory genes enable us to design effective strategies for improving soybean seed qualities and production. The research is supported by USDA-ARS and the United Soybean Board.

Poster Number: 5 Evolution of Recognition Specificity in a Cluster of Soybean Disease Resistance Genes

Tom Ashfield, Indiana University, Bloomington Thomas Redditt, Indiana University, Bloomington Andrew Russell, Indiana University, Bloomington Ryan Kessens, Indiana University, Bloomington Qing Kang, Lauren Galloway, Natalie Rodibaugh, Roger Innes

Effector triggered immunity in plants involves highly specific recognition events in which plant resistance (R) proteins either detect pathogen effector proteins directly, or alternatively, the modifications that they inflict on the host cell. The molecular basis of recognition specificity, and how new specificities evolve, remain important questions. We are addressing these questions by studying two closely linked soybean genes, Rpg1b and Rpg1r, which mediate detection of the Pseudomonas syringae effector proteins, AvrB and AvrRpm1, respectively. At least Rpg1b appears to detect its target effector indirectly, probably by monitoring modification of soybean homologues of the Arabidopsis RIN4 protein. We have previously cloned Rpg1b and have now identified a strong candidate for Rpg1r, both of which are CC-NB-LRR type genes. Rpg1b and Rpg1r are most similar over a stretch that includes the second half of the CC and the complete NB-ARC domain, with the N-terminal half of the CC domain and the LRR domain being significantly less similar. This similarity pattern is likely the result of a gene conversion event that introduced the NB-ARC region from Rpg1r, or a closely related paralogue, into the ancestral Rpg1b lineage. The tertiary structures of the Rpg1b CC, NB-ARC, and LRR domains have been modeled based on comparison to known structures. Superimposition of the polymorphisms between the Rpg1b and Rpg1r amino acid sequences onto these structures reveals highly polymorphic surface regions in both the CC and LRR domains, suggestive of a role for these regions in influencing Rpg1 R protein specificity. Recognition of AvrB and AvrRpm1 in Arabidopsis depends on a second plant protein, RIN4, which is phosphorylated in the presence of the pathogen effectors. Four RIN4 homologues (gmRIN4s) have been identified in soybean. All four gmRIN4s are cleavable by AvrRpt2, which also strongly suppresses AvrB and AvrRpm1 recognition in this species. Interestingly, preliminary results indicate that gmRIN4B interacts weakly with both the Rpg1b and Rpg1r CC domains in yeast.

Poster Number: 6 Soybean SNP Discovery for Important Traits through Next Generation Sequencing (NGS) at Dow AgroSciences

Yonghe Bai, Dow AgroScience LLC Tyler Mansfield, Fang Lu, Jenelle Meyer, Clive Evans, Shrinivasrao, Mane, Steve Rounsley, Evelyn Ortiz-Perez, Hunt Wiley, Brad Hedges, Jan Erik Backlund, David Meyer and Steve Thompson

Next generation sequencing (NGS) technologies are becoming very powerful tools for SNP discovery, providing new ways to accelerate fine-mapping and gene isolation in many species. The throughput for NGS is often measured in billions of base pairs per run - orders of magnitude increase over traditional Sanger sequencing. Tremendous progress in the field of genomics for many crops has been made in a short period due to the rapid development of NGS technologies and the resulting increase in throughput and reduction in cost. We applied whole genome sequencing to discover new SNPs among selected DAS soybean cyst nematode (SCN) resistant and susceptible lines. Over 50 million reads were generated for each of the samples using the Illumina HiSeq 2000 platform (Illumina®). There are a total of 208,885 and 45,150 significant SNPs at P value <=0.05 and P value <=0.01, respectively. Of these SNPS, 28,619 and 8,330 were present in genes (at P value <=0.05 and <=0.01, respectively). The SNP distribution across 20 chromosomes is not even. For validation, we created KASP (KBioSciences, England) assays for a selected sub-set of SNPs and genotyped the same samples that had been sequenced for SNP discovery. The percentage of true SNPs is above 90%. Some of the SNP selection criteria and tools used to analyze the SNP data in this project will be also discussed.

Poster Number: 7 SNP Discovery Pipeline for Utilization of NGS Data in Soybean Breeding Program

Carmille Bales, Michigan State University Dechun Wang, Michigan State University

Marker-assisted selection by single nucleotide polymorphism (SNP) assay genotyping has been very valuable in plant breeding programs in recent years owing to its abundance, robustness and relatively low cost. For high sample throughput application in soybeans, KBiosciences' KASPar and TaqMan® custom-designed SNP genotyping assays allow genotyping of thousands of breeding lines for a single or up to 6- multiplexed markers. The custom-designed assays are ideal for screening fine mapping populations or a large F2 population for one or some target traits. SNP discovery, however, for a small breeding lab can be the bottleneck in the whole process which involves cloning of PCR fragments, re-sequencing and validation using SNP detection methods.

Here we present a general pipeline that can be used for SNP mining of available and future whole genome re-sequencing projects of soybean accessions that represent a core set of parental lines in a breeding program. In our lab, twelve soybean accessions that are used as parental lines for soybean aphid resistance breeding and high yield selections were re-sequenced to at least five read-depth coverage on the Illumina HiSeq 2000 platform at $782 per accession. A total of about 62 to 101 million paired- end reads of 100-bp length were used to align short reads into the soybean reference genome, Williams 82 (Glyma 1.09 assembly). After read quality assessment, the Bowtie software was used as an efficient tool for read mapping and SNP consensus calls were generated using SAMTools. Using open source quality-aware programs such as Bowtie and SAMTools, a process flow for bioinformatics analysis was established for efficient SNP calling of the re-sequenced lines. The identified SNPs among parental lines were aligned to each other using visualization tools and used to identify polymorphic SNP markers of higher resolution for bi-parental populations that were otherwise undetected using the Illumina SoySNP50 Beadchip assay.

Validation of computationally identified SNPs was done by designing KASPar assays to genotype a subset of lines representing a fine mapping population. Within an interval of 230-kb genomic region, 100 SNPs that were polymorphic between a resistant (PI 567598B) and susceptible line (Skylla) were identified to have at least 5x coverage and high quality call scores. Out of these, 10 SNPs were selected at 25-kb distance per SNP to identify recombination breakpoints that can narrow a genomic region correlated to soybean aphid resistance. Experiments are still ongoing for the validation of polymorphic SNPs.

Poster Number: 8 Identification and Introgression of Novel Sources of Resisance to Soybean Cyst Nematode

Yong Bao, University of Minnesota Lian Lian, James Orf, Senyu Chen, Nevin Young, and Roxanne Denny

Soybean cyst nematode (SCN, Heterodera glycines Ichinohe) is the most yield-limiting pathogen on soybean. Genetic resistance is the most cost-effective and environmentally friendly method to control SCN. The main purpose of this study is to characterize SCN resistant sources that differ from Peking or PI 88788, targeting PI 567516C, to identify and map QTL and to develop SNP markers for use in marker-assisted backcross selection (MABS). QTL mapping was performed with the 184 F2:3 progeny derived from a MN0095 (susceptible) x PI 567516C (resistant) cross. DNA was extracted from one young leaf of each F2 plant using DNeasy 96 Plant Kit (QIAGEN) and the 1536 SNP markers in Universal Soy Linkage Panel (USLP 1.0) was used for genotyping the F2 plants and both parents. Genotyping was performed with Illumina GoldenGate Assay in two 96 well plates. Non-parametric QTL mapping methods and Composite interval mapping (CIM) methods were performed using R/qtl to map QTL. A permutation test was performed with 1,000 runs to determine the P=0.05 genome-wide significance level for declaring a significant QTL. Two significant QTLs (genome-wide type I error =0.05) and two suggestive QTLs (LOD > 3) were declared as associated with resistant to SCN HG Type 2.5.7 and HG Type 1.2.5.7. The two significant QTLs were detected on chromosome 10 and chromosome 19 and the two suggestive QTLs were detected on chromosome 8 and chromosome 18 (locate at or near Rhg1). Computational analysis was conducted in the genomic region of major QTL with the greatest LOD score located on chromosome 10 for gene ontology and function grouping. SNP markers are being developed and validated to increase marker density on both sides of the chromosome 10 QTL peak, and will be implemented in MABS for QTL introgression. Selected F2 and F4 plants from the same cross with resistant markers will be backcrossed with the recurrent parent MN0095 for three generations. Agronomic evaluation and assay of SNP markers will be applied in every cycle of backcross to achieve an ideal genotype. SCN bioassays will also be performed in greenhouse to confirm the introgression of the novel QTL with resistance to SCN multiple HG types in backcross populations. This study suggests that QTL mapping and MABS with SNP markers can facilitate the delivery of superior germplasm with novel SCN resistance in soybean.

Poster Number: 9 Is Carbonic Anhydrase the Target of FvTox1 Which Initiates Foliar SDS?

Jordan Baumbach, Department of Agronomy, Iowa State University, Ames, Iowa 50011 Ramesh Pudake, Department of Agronomy, Iowa State University, Ames, Iowa 50011 Sivakumar Swaminathan, Department of Agronomy, Iowa State University, Ames, Iowa 50011 Madan Bhattacharyya, Department of Agronomy, Iowa State University, Ames, Iowa 50011

Fusarium virguliforme causes sudden death syndrome (SDS) in soybean. The estimated average annual yield suppression from this disease has been valued over 100 million dollars. The foliar symptoms of the disease includes chlorosis, necrosis and in severe cases defoliation and sudden death. F. virguliforme is a root pathogen and has never been isolated from the above ground foliar tissues that show disease symptoms. F. virguliforme produces an acidic proteinaceous toxin, FvTox1, involved in foliar disease symptom development. FvTox1 was localized to chloroplasts. Most likely the toxin molecules interact with soybean protein(s) to impair a vital function in soybean. To identify such protein(s) we applied a yeast two-hybrid screening and isolated 29 soybean proteins, many of which are localized to chloroplasts. In order to narrow down the list of candidate FvTox1-interacting proteins we determined the single nucleotide polymorphisms of the isolated genes from the susceptible cultivar, Essex, with those from the moderately resistant cultivar, Williams 82. A carbonic acid anhydrase gene showed polymorphisms for seven nucleic acid residues. Four of these polymorphisms were non-synonymous. Two non-synonymous changes were located in a putative motif that is involved in situ interaction with the central domain of FvTox1. The interaction of FvTox1 was almost absent with the maize homologue of the protein. Transgenic soybean plants expressing the maize homologue of the gene were much more tolerant to FvTox1 as compared to the non-transgenic control. This study suggests that most likely FvTox1 interferes with CO2 import into chloroplasts for photosynthesis by interacting with a carbonic acid anhydrase isoform in order to initiate foliar SDS.

Poster Number: 10 Genome-Wide Sequence Analysis and Expression Profiling Using RNA-Seq Implicates Soybean (Glycine max) HD-Zip Gene Family Members in Dehydration and Salt Stress Responses

Vikas Belamkar, Iowa State University, Ames, Iowa, 50011, USA Nathan Weeks, USDA-ARS CICGRU, Iowa State University, Ames, Iowa, 50011, USA Arvind Bharti, National Center for Genome Resources, Santa Fe, New Mexico, 87505, USA Andrew Farmer, National Center for Genome Resources, Santa Fe, New Mexico, 87505 USA Michelle Graham, USDA-ARS CICGRU, Iowa State University, Ames, 50011, Iowa, USA; Steven Cannon, USDA-ARS CICGRU, Iowa State University, Ames, 50011, Iowa, USA

Homeodomain leucine zipper (HD-Zip) transcription factors are unique to plants, with diverse roles in growth, development, and response to environmental stress. HD-Zip genes have been well characterized in Arabidopsis thaliana, but are yet to be elucidated in soybean. In this study we identify all members of the HD-Zip gene family in soybean, and characterize their expression under dehydration and salt stress. Homology searches identify 101 HD-Zip genes in the soybean genome. Phylogeny reconstruction coupled with domain and gene structure analyses using soybean, Arabidopsis, rice, grape, and Medicago homologues enables placement of these sequences into four previously described subfamilies. HD-Zip genes are distributed over all twenty soybean chromosomes. Of the 101 HD-Zip genes, 96 exist as paralogous pairs, with the duplicates having being retained following paleopolyploidy events. An RNA-Seq experiment performed to study differential gene expression at 0, 1, 6 and 12 hr in the roots of soybean cv. ‘Williams 82’ under dehydration and salt stress identifies 22 differentially expressed genes (DEGs). Several of these DEGs are located in QTLs previously reported for yield-related traits under abiotic stress in soybean. Screening of HD-Zip gene promoters identifies numerous cis-elements that are overrepresented in the promoters of DEGs in soybean under both stresses, consistent with the role of HD- Zip in abiotic stress responses. A candidate gene hypothesized to be involved in leaf nastic responses under water stress has been selected for functional characterization.

Poster Number: 11 Evaluating Soybean Aphid Resistance in Exotic Soybean Lines

Carolyn Bonin, University of Illinois, Urbana-Champaign Curtis Hill, University of Illinois Glen Hartman, USDA-ARS, University of Illinois Brian Diers, University of Illinois

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is a common insect pest of soybean [Glycine max (L.) Merr] in China and also is found frequently in other Asian countries. It was identified in the US in 2000 and has since spread throughout the soybean growing regions of the US and Canada. There are currently four known soybean aphid resistance genes (Rag1 through Rag4) approved by the Soybean Genetics Committee. Soybean aphid biotypes have been identified that are able to overcome Rag1 and Rag2 even prior to the wide-spread production of cultivars with these two genes. The objective of this research was to study the genetic basis of aphid resistance in plant introductions (PIs) with a goal of identifying new aphid resistance genes.

Soybean aphid resistance was examined in the greenhouse in a collection of F2 families developed from 19 soybean aphid resistant PIs. Based on greenhouse segregation ratios of aphid resistant and susceptible responses, resistance appeared to be controlled by a single dominant gene in 14 populations, two genes in one population, a single recessive gene in one population, and three populations had no clear Mendelian segregation ratio. Markers closely linked to Rag2 were significantly associated with resistance in 14 of the populations, indicating that this locus is important in soybean aphid resistance. Markers linked to Rag1 were significantly associated with resistance in five populations and Rag3 was significantly associated with resistance in one population. Ten of the populations also were genotyped by bulk segregant analysis using the GoldenGate assay and these results largely confirmed the single marker results. From this study, we conclude that soybean aphid resistance in PIs is frequently controlled by one or two genes, and the Rag2 locus is often associated with resistance.

Poster Number: 12 Previously Identified Insect Resistance QTLs Are Effective Against the Kudzu Bug

Adam Bray, University of Georgia John N. All, H. Roger Boerma, Zenglu Li, Wayne A. Parrott

The kudzu bug (Megacopta cribraria) is the most recent insect infesting soybean. It was first detected in Georgia in the fall of 2009, and is thought to have migrated from Asia through the Atlanta airport. It has rapidly spread throughout Georgia, and into adjacent states. In China and India, this insect feeds on kudzu, soybean, and other legumes, causing yield losses of up to 50%. Survey data from Georgia reveals infestations of 500 kudzu bugs per plant if soybeans are left untreated.

Over the past two decades, the soybean genes for resistance to leaf-chewing insects have been characterized. Specifically, three QTLs: QTL-M, QTL-G, and QTL-H from the Japanese land race PI229358 provide resistance to a broad range of leaf-chewing lepidopterans and coleopterans. QTL-M acts via both antibiosis and antixenosis, while QTL-G is antibiotic and QTL-H is antixenotic. Importantly, QTLs G and H are effective against lepidopterans only if the resistant allele for QTL M is also present. Additional resistance is conferred by sharp-tipped trichomes. All these sources of resistance to leaf-chewing insects were evaluated for their effectiveness against the kudzu bug. We conducted field tests in one location in 2010, and two locations in 2011. Soybean isogenic lines for QTL-G, and QTL-H showed reduced insect infestations; and a trend for less yield loss in QTL-H soybean compared to the other isolines was also observed. In contrast to lepidopterans, QTL-M was not effective against the kudzu bug, nor was its presence required for QTL-H and QTL-G to be effective. Likewise, sharp trichomes were not effective against the kudzu bug.

The kudzu bug, a hemipteran, represents a third order of insects that can be controlled with the known QTLs. Furthermore, the kudzu bug is not a leaf-chewer. The ability of single genes to confer resistance against such a range of insects is unprecedented; therefore, efforts are underway to fine-map and clone QTLs G and H, in order to obtain a better understanding of the biochemistry of resistance.

Poster Number: 13 Functional Analyses of the Candidate Fusarium virguliforme Genes Up-Regulated during Infection of Soybean Roots

Catherine Brooke, Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Jordan Baumbach, Department of Agronomy, Iowa State University, Ames, IA 50011 Binod Sahu, Department of Agronomy, Iowa State University, Ames, IA 50011 Ramesh Pudake, Department of Agronomy, Iowa State University, Ames, IA 50011 Madan K. Bhattacharyya,Department of Agronomy, Iowa State University, Ames, IA 50011

The estimated average annual U.S. soybean yield loss from the fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome (SDS), is estimated to be over $100 million. Due to this economic and agricultural impact, it is becoming urgent to create soybean germplasm with novel SDS resistance by applying biotechnological approaches. To facilitate development of such germplasm, we must have a better understanding of the mechanisms used by F. virguliforme to cause SDS. To accomplish this, the transcriptome of infected soybean roots was analyzed and 377 genes were identified as being induced during infection. These genes are involved in processes such as: synthesis of polyketide-type of toxins, establishment of localization, localization, transmembrane transport, transport mechanisms, cellular process, metabolism, transporter activity, oxidoreductase activity, cation binding, and hydrolase activity. We have applied a homologous recombination approach to knock-out genes that are highly up-regulated during infection of soybean. To date, we have identified mutants of 14 candidate F. virguliforme pathogenicity genes. Knockout mutants were confirmed by PCR and are being analyzed for possible virulence and other pathogenicity and phenotypic characteristics.

Poster Number: 14 Identification of Senescence Associated Genes in Glycine max Cotyledons

Anne Brown, Purdue University

Immediately following germination, the developing soybean plant relies on the nutrient reserves stored in the cotyledons to sustain heterotrophic growth. Subsequently, cotyledons develop into the first photosynthetically active tissue of the growing soybean with a defined life span of about three weeks. The viability of these tissues during this period is crucial to seedling establishment. Before the cotyledon senesces, the nutrients must be mobilized and transported to newer parts of the plant. To gain insights into these specialized leaves, we used RNASeq to compare levels of gene expression throughout cotyledon development in Glycine max. Over 48,000 transcripts were found to be expressed during the course of cotyledon development, 47.9% of these genes show significant differential expression between at least two of the stages examined. The alpha/beta-hydrolase super family were one of many shown to be differentially expressed throughout the stages. Genes significantly up-regulated in stage two functioned in proteolysis and peptidolysis, while genes significantly up-regulated in stage three functioned primarily in lipid metabolism. We present a comparison of genetic regulation of cotyledon senescence to previous studies done on leaf senescence in Arabidopsis.

Poster Number: 15 The Legume Information System 2012

Steven Cannon, USDA-Agricultural Research Service Andrew Farmer, NCGR Arvind Bharti, NCGR Ken Seal, NCGR Nathan Weeks, Benjamin Mulaosmanovic, John Crow, Rex Nelson, David Grant, Randy Shoemaker

The mission of the Legume Information System (LIS) is to facilitate discoveries and crop improvement in the legumes, which are critical components of global food and agriculture systems. LIS stores datasets from numerous legume species, and uses the reference species Glycine max, Lotus japonicus, and Medicago truncatula as a basis for comparisons between and among diverse legume species. Genomic references like pigeonpea (recently published) and others will also be added as they become available. For other legume species, LIS hosts transcriptome assemblies and other datasets. Comparative maps, reference datasets, sequence search tools, etc. make these datasets available for exploration and discovery. A new site layout offers new species pages, as well as a number of entry points for exploration by gene, trait, function, and genomic context. The LIS multi-sequence, multi- target search tool, Seqqle, serves as a portal to genome browsers at SoyBase, the JCVI Medicago truncatula Project, the Medicago HapMap Project, and Kazusa's Lotus japonicus site, and to the Medicago truncatula Gene Expression Atlas at the Noble Foundation. LIS is funded by the USDA- ARS, and is developed and maintained jointly by the National Center for Genome Resources (NCGR) and the USDA-ARS at Ames, Iowa. The team invites you to explore LIS at http://comparative-legumes.org and welcomes your comments and suggestions.

Poster Number: 16 Functional Analyses of Soybean Matrix Metalloproteinase Genes

Doris Carbajulca, University of Illinois at Urbana-Champaign David Neece, USDA-ARS Steve Clough, USDA-ARS, University of Illinois at Urbana-Champaign

Matrix metalloproteinase (MMPs) are metal-dependent proteases involved in the degradation of extracellular matrix substrates and in the remodeling of the matrix. When some substrate proteins are degraded, they release biologically active peptides that serve as signaling molecules. The exact role of MMPs in plants is not fully understood. However, it has been suggested that plant MMPs play crucial roles in plant development, senescence, and responses to biotic and abiotic stresses. Microarray analyses in our lab revealed that several soybean MMPs are induced fairly specifically by pathogens. Searching the soybean genome (http://www.phytozome.net/soybean) we identified a total of 27 MMP homologs. We designed primers to six of these MMP genes to examine specific gene expression in response to pathogens, using qRT-PCR analysis. We identified three MMP genes that were differentially induced in soybean plants challenged with Pseudomonas syringae, Sclerotinia sclerotiorum, and Fusarium virguliforme, suggesting that these genes might play a role in soybean general response to pathogens. Virus Induced Gene Silencing (VIGS) is a method that allows for elucidation of gene function, and we used a VIGS vector system based on Bean Pod Mottle Virus (BPMV) to develop constructs for silencing MMP genes in soybean. The soybean MMP gene family presents a challenge for VIGS studies, however, due to high homology among family members. We designed VIGS constructs for silencing individual family members, as well as constructs for silencing multiple MMP genes. Experiments are currently underway to assess these constructs in soybean. MMP function is also being investigated by overexpression studies in Arabidopsis and eventually soybean.

Poster Number: 17 Wild Soybean (Glycine soja); An Excellent Genetic Resource for Increasing Omega-3 fatty Acid (Linolenic Acid) in Cultivated Soybean

Jong-Hyun Chae, School of Applied Biosciences, Kyungpook National University, Republic of Korea Sovetgul Asekova, School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea, Ju-Eun Pakr, School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea, Bo-Keun Ha, School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea, Cuyhwa Chung, Department of Biotechnology, Chonnam National University, Yeosu, Republic of Korea; J. Grover Shannon; Divison of Plant Sciences, Universtiy of Missouri-Delta Center, Portageville, MO 63873, USA; Jeong-Dong Lee: School of Applied Biosciences, Kyungpook National University, Daegu 702-701, Republic of Korea

Scientific studies have shown that essential fatty acid intake can have a dramatic impact on human health. Soybean [Glycine max (L.) Merr.] oil from current commercial cultivars typically contains around 8% linolenic acid (18:3) known as omega-3 fatty acid. Omega- 3 fatty acid plays an important role to prevent cardiovascular disease and cancer. Relatively high 18:3 content in seed oil is a trait of the wild soybean (Glycine soja Sieb. and Zucc.) ancestor of modern soybean cultivars. Wild soybean is native to Korean peninsula and recently thousands of wild soybeans collected by soybean researchers in Korea. The objective of this study were to determine the linolenic acid content for wild soybean collection and to determine the stability of linolenic acid content derived from wild soybean over environments. Fatty acid profile for 1,806 wild soybean accessions collected from South Korea was determined by GC. The range of linolenic acid was 7.3 to 23.7% with an average 15.6%. We developed a recombinant inbred population from a cross PI483463 (wild soybean with 15% 18:3) and Hutcheson (cultivar with 8% 18:3). Three RILs, RIL156, RIL159 and RIL166, with high linolenic acid content (over 14%), parents and Williams 82 as checks were grown in nine environments over 2008-2011. Results showed that the content of linolenic acid for the PI483463, Hutcheson, and Williams 82 ranged from 14.8 to 17.1, 8.5 to 9.7, and 6.9 to 8.4 % and averaged 15.4, 9.2 and 8.0%, respectively. However selected RILs 156, 159, and 166 ranged from 10.7 to 15.7, 14 to 15.8, and 14.8 to 15.8, and averaged 13.9, 14.9, and 15.2, respectively. Among the tested accessions, RIL166 was the most stable with the lowest range and CV, and had a relatively lower stability coefficient value than other genotypes. Genes related to high linolenic acid from wild soybean may be useful in developing higher linolenic acid soybean genotypes and would broaden the use of soybean in food applications to improve human nutrition and health. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education Science and Technology (#2010-0003220). *Corresponding author: Tel. 053-950-5709, E-mail: [email protected] Poster Number: 18 Transcription Factor ORFeome for Building Genetic Regulatory Networks Associated with Drought Resistance in Soybean

Chenglin Chai National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO, 65211 Yongqin Wang National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO, 65211 Babu Valliyodan National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO, 65211 Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO, 65211

Soybean (Glycine max L.) is an important source of protein for human and animal nutrition, as well as a major source of vegetable oil. Soybean growth is affected by unfavorable environmental factors including drought, salinity, and high/low temperature which significantly decrease soybean yield worldwide. Therefore, future soybean production will greatly depend on scientific advances which allow soybean producers to remain economically competitive. In this study, emerging Omics techniques will be employed for building genomic resources and to dissect drought resistance regulatory networks in soybean.

We are building soybean genomic resources through the isolation and cloning of transcription factor (TF) full-length open reading frames (ORFs), focusing on factors likely involved in the regulation of genes associated with abiotic stress responses. Gateway vectors were adopted for high-throughput cloning and functional studies. Downstream studies include genome-wide mapping of interactions between protein- protein and protein-DNA using various tools such as Yeast Two-Hybrid (Y2H) and Chromatin Immuno-precipitation followed by high through-put sequencing (ChIP-Seq) and global gene expression profiling (RNA-Seq). We have cloned around 100 ORFs into pENTRTM/D TOPO vectors and the project target for the first phase will be 300 TFs- ORFs. We have generated antibodies for selected soybean TFs and the immuno- precipitation and ChIP-Seq experiments are in progress. Also, transgenic Arabidopsis plants with 10 gene constructs were developed and the in-planta functional characterization of these TFs is in progress.

Poster Number: 19 Improvement of Michigan-Bred Soybean Aphid-Resistant Lines by Combining Aphid- Resistance Genes with Defoliation-Resistance to Japanese Beetle

Desmi Chandrasena Crop & Soil Sciences, Michigan State University Dechun Wang Crop & Soil Sciences, Michigan State University Chris DiFonzo Entomology, Michigan State University

The soybean aphid, Aphis glycines Matsumura (Hemiptera: Aphididae), is native to Asia, and is a major destructive pest of soybean Glycine max (L.) Merr. Up to 50% yield loss can occur in the United States if fields are left untreated. Several aphid-resistant soybean lines have been developed by many breeding programs, but only very few lines were successful due to breakdown of resistance. Thus far, the aphid-resistant lines developed by the MSU (Michigan State University) soybean breeding program continue to show reliable resistance. During a field and laboratory assessment conducted through 2007-2010, the MSU aphid-resistant lines E06901, E06905, and E06906 showed elevated susceptibility to defoliation (>50% in field choice tests) by Japanese beetle (JB) Popillia japonica Newman, when compared with another aphid-resistant line (LD05-16060) developed by the Uni. of Illinois. Typically action thresholds for soybean defoliation range from 30%-40% pre-bloom, decreasing to 15% between bloom and pod fill, and 25% thereafter. This issue led to identification of new lines among aphid- resistant germplasm that also show resistance to JB defoliation. A QTL (Quantitative Trait Loci) mapping approach was used to discover QTLs linked to JB resistance in a population derived from E06906 x LD05-16060. Pest severity (PS) and Pest Incidence (PI) data were collected for two years on the same study site in East Lansing. Preliminary mapping with 94 individuals on 13 chromosomes showed putative QTL peaks on several positions; some corresponding to known major QTL. Further research on this population identified a potential QTL for Japanese beetle resistance on chromosome 5. Key candidate genes involved in the flavonoid biosynthesis pathway were found within this potential QTL region. Thus, flavonoids may play a key role in explaining differential defoliation by JB on these aphid-resistant lines. Furthermore, metabolite profiling is underway to investigate the differences in key biochemical compounds between these JB-resistant and JB-susceptible lines. Finally, this new direction also provided an opportunity to stack aphid-resistant genes (Rag1 with rag3 and rag1b), thereby improve the MSU aphid-resistant germplasm to confer strong resistance to both soybean aphid and JB.

Poster Number: 20 Identification of High Quality Single Nucleotide Polymorphisms in Glycine latifolia using the G. max Genome Sequence as a Reference

Sungyul Chang Department of Crop Sciences, University of Illinois Glen Hartman USDA-ARS, Department of Crop Sciences, University of Illinois Ram Singh Department of Crop Sciences, University of Illinois Kris Lambert Department of Crop Sciences, University of Illinois Houston Hobbs, Department of Crop Sciences, University of Illinois; Leslie Domier, USDA-ARS, Department of Crop Sciences, University of Illinois

Like many widely cultivated crops, soybean [Glycine max (L.) Merr.] has a relatively narrow genetic base, while its 26 distant perennial relatives in the subgenus Glycine Willd. are more genetically diverse and display desirable traits not present in cultivated soybean. For example, high levels of resistance to Sclerotinia sclerotiorum, which causes Sclerotinia stem rot, have been identified in G. latifolia and other perennial wild Glycine species, even though only low levels of resistance have been identified in G. max. To generate molecular resources for gene mapping and identification in a perennial Glycine species, single nucleotide polymorphisms (SNPs) were identified in G. latifolia by high-throughput sequencing of reduced-representation libraries prepared from DNAs from accessions PI 559298 (resistant) and PI 559300 (susceptible). Approximately 30% of the 36 million 100-nt reads produced from each accession aligned primarily to gene-rich (euchromatic) regions on the distal arms of G. max chromosomes. Because a genome sequence is not available for G. latifolia, the G. max genome sequence was used as a reference to locate G. latifolia single nucleotide polymorphisms (SNPs), which identified an average of 450 SNP-containing sequences that aligned to each of the 20 G. max chromosomes. To test the usefulness of the SNPs and the synteny between the G. latifolia and G. max genomes, nine allele-specific assays were designed from polymorphic sequences that aligned to G. max chromosomes 4 or 13. All nine markers segregated in the expected 1:2:1 ratio in a G. latifolia F2 population, formed two distinct linkage groups, mapped in similar orders in G. latifolia and G. max, and differentiated homoeologous loci. These results showed that a heterologous reference genome sequence can be used to identify informative high-quality SNPs in a related species.

Poster Number: 21 Approaches To Enhance Photosynthetic Capacity of Soybean

Timothy Changa Center for Plant Science Innovation, University of Nebraska-Lincoln Saadia Bihmidine, Donald Weeks, Tala Awada and Thomas Clemente

Enhancing the photosynthetic capacity of C3 crops such as soybean may improve yield potential and/or protect yield under biotic or abiotic stress conditions. As a mean to explore this avenue of work we introduced into soybean two cyanobacterial genes that previously were shown to enhance photosynthesis and growth in plants. To this end we inserted constitutively expressing cassettes carrying an inorganic carbon transporter B (ictB) along with a dual fructose-1,6-/sedoheptulose-1,7-bisphosphatase (FBPase/SBPase) genes from Synechococcus sp. Selected transgenic soybean events were phenotyped under both greenhouse and field conditions. Under greenhouse conditions, the ictB events showed increase in both net photosynthesis (Anet) and instantaneous water use efficiency (iWUE) compared to the wild types. On the other hand, we did not record any significant changes in stomatal conductance (gs), suggesting that the observed increase in Anet was most likely due to the increased concentration of CO2 at the site of Rubisco. Although, the greenhouse results were confirmed under field conditions, the increase in Anet in the field did not translate into an increase in yield. With regards to the FBPase/SBPase transgenic events, our results showed that these genes, while they had some positive impacts on Anet under both greenhouse and field conditions, results were variable and not as promising as those observed with the ictB events. Building upon these findings we subsequently redesigned a set of genetic cassettes consisting of soybean codon-optimized versions of ictB and a tomato SBPase, placed under control of the pea rbcs promoter, individually and stacked within the same T-DNA element. These have been introduced into soybean and transgenic events are currently being characterized at the molecular level.

Poster Number: 22 ABA Perception and Signaling in Soybean

Wei Chen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri Columbia, MO 65211 Mingzhe Zhao National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri Columbia, MO 65211 Babu Valliyodan National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri Columbia, MO 65211 Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri Columbia, MO 65211

In higher plants, abscisic acid (ABA) is a key endogenous phytohormone which triggers plants’ stress response under biotic and abiotic environmental challenges such as; pathogen attack, cold, and drought. Understanding the molecular mechanisms underlying ABA perception and signaling can provide valuable information for improving plant performances under stress conditions. Recent breakthroughs in the field of ABA signaling have uncovered novel class of ABA receptors (Pyrabactin Resistance (PYR)/PYR-Like (PYL) receptors) which plays key role in plant cell signaling cascades. Binding of ABA to the PYLs inactivates type 2C protein phosphatases (PP2Cs) leading to the activation of SNF1-related protein kinase 2 (SnRK2), which targets ABA- dependent gene expression. We used soybean as a model to study the ABA perception and signaling cascades in legume crops. The soybean genome encodes at least seventeen PYL receptors, seven PP2Cs and five SnRKs. Phylogenetic and transcriptomic analyses have revealed that there are two major clades of PYLs: one clade of six members abundantly expressed in the whole plant; and the other clade containing eleven root-specifically-expressed members. We have performed a stringent binary yeast two-hybrid matrix (43X43) experiment to map protein-protein interactions between ABA signaling components and found that PYL-PP2C and PP2C-SnRK interactions are conserved in soybean. We are currently profiling the expression of various PYL/PP2C/SnRK proteins in different tissues in the presence of exogenic ABA and under stress conditions such as high salt concentration, cold, and drought. Transgenic Arabidopsis plants over-expressing ten PYL receptors have been generated and will be used to characterize differential functionalities among tissue-specific ABA receptors. These analyses will guide us by manipulating central players in ABA perception and signaling cascades to improve soybean performance under biotic and abiotic stresses.

Poster Number: 23 Genetics and Mapping of Virulence in the Soybean Aphid

Anitha Chirumamilla University of Illinois Rosanna Giordano University of Illinois Curt Hill University of Illinois Glen Hartman USDA-ARS/University of Illinois

Aphis glycines Matsumura (Hemiptera: Aphididae), the soybean aphid, is the most significant insect pest of soybean [Glycine max (L.) Merr.] with an economic impact on annual soybean production in North America estimated at US$3.6 to $4.9 billion. Several soybean aphid resistance genes were identified and mapped in soybean. However, before this resistance could be deployed into production, soybean aphid biotypes with the ability to overcome known resistance genes were discovered. This discovery indicated the existence of diverse virulence in soybean aphid populations in North America and a high potential for rapid adaption to resistance genes, especially those expressing antibiosis-type resistance. Deployment of soybean cultivars with soybean aphid resistance is a major component of IPM. Knowledge of the diversity and geographic distribution of aphid biotypes in soybean production regions will help optimize the use of resistance with a strategy of intelligent deployment of resistance genes into production areas with a low potential for soybean aphid adaptation to the resistance genes. Targeted deployment of resistance may increase the effectiveness of host resistance and retard aphid evolution toward Rag gene adaptation, thus increasing the durability of resistance. However, intelligent deployment of resistance gene(s) is dependent upon accurate monitoring of virulence in soybean aphid populations. One way to monitor soybean aphid virulence is screening samples of aphids on a set of differential soybean lines. More cost effective and time efficient monitoring of soybean aphid virulence would employ DNA markers closely associated with aphid virulence to screen the soybean aphid populations. To determine the genetics of soybean aphid virulence, three documented biotypes (1, 2, and 3) have been crossed and mapping populations are in development, the transcriptomes of the three biotypes and the biotype 1 genome have been sequenced, numerous SNPs and microsatellites have been identified from the sequence information and markers are under development. Aphid mapping populations that have been phenotyped for virulence on specific Rag genes will be screened with markers that are polymorphic between the biotypes to identify markers associated with virulence.

Poster Number: 24 Endogenous siRNAs that Silence Chalcone Synthase Result in Pigment Pattern Formation on Seed Coats in Glycine max

Young Cho University of Illinois, Crop Sciences, Urbana, IL, 61801 Lila Vodkin University of Illinois, Crop Sciences, Urbana, IL, 61801

In soybean, dominant alleles of the I locus inhibit pigmentation of the seed coat while the homozygous recessive i allele results in fully pigmented seed. Previous work showed that dominant alleles correspond to duplications of chalcone synthase (CHS) genes leading to production of CHS siRNAs which in turn degrade CHS mRNAs resulting in yellow seed coats (Tuteja et al. Plant Cell 21, 2009). However, it was not known if the same phenomenon applied to the yellow and pigmented regions within the same seed coats that have homozygous i-i or i-k alleles that restrict pigment to the hilum and saddle regions of the seed coat, respectively. Here we describe the results of Illumina high throughput sequencing of small RNA populations from pigmented and yellow regions within seed coats with the same genotype. The level of CHS siRNAs is much higher in the yellow versus the pigmented region. CHS siRNAs from another genotype (i-i, k1), which also produces a pigmented saddle on the seed coat, are also much more abundant in the yellow versus the pigmented region. Small RNA blots show CHS siRNAs accumulated only in the yellow region and confirm the sequencing data. Thus, these data demonstrate that CHS siRNAs result in pigment pattern formations on soybean seed coats. In addition, small RNA sequencing data from twenty-one samples in six different tissues including cotyledons, roots, stems, and leaves showed that CHS siRNA expression is limited to the yellow seed coats confirming the tissue specificity of the generation of the CHS siRNAs. We gratefully acknowledge support from the USDA, United Soybean Board, and Illinois Soybean Association.

Poster Number: 25 Identification And Characterization of Novel Micro Rnas from Soybean Leaves Infected by Pseudomonas syringae pv. glycinea

Steven Clough USDA-ARS Osman Radwan University of Illinois Bernarda Calla University of Illinois Lila Vodkin University of Illinois

Bacterial effector proteins, secreted through type III secretion systems, have been shown to trigger defense responses when recognized by resistant plants, and to suppress defense responses in susceptible host plants. Our results of gene expression profiling indicated that while many defense related genes and signal transduction components were induced in the incompatible reaction, they were suppressed in susceptible plants inoculated with the same avriulent strain of Pseudomonas syringae (proposed savastanoi) v. glycinea (Psg) carrying avrB.

Here, we examined the possible role of samll RNAs (smRNAs) as a mechanism by which host genes are regulated during soybean-Psg interactions. A total of 4,231,029 and 3,740,768 smRNA reads from Williams 82 (resistant) and Flambeau (susceptible), respectively were obtained from deep sequencing. Out of unique signatures of 34,171 smRNAs, 51 novel microRNAs (miRNAs) were identified (0.15%). Most of these novel miRNAs perfectly matched 1 or 2 locations of soybean genome and derived from intergenic regions of soybean genome.

Expression data from these novel miRNAs as a ratio between infected resistant and susceptible plants revealed that 28 miRNAs were up regulated with log2 >= 1.5 and 21 miRNAs decreased in expression levels log2 <= -1.5. Additionally, soybean putative targeted genes of these novel miRNAs have been identified. Results from Northern blots of some of these novel miRNAs and qRT-PCR of targeted transcripts will be presented.

Poster Number: 26 Transposon Mutagenesis in Soybean

Yaya Cui University of Missouri-Columbia Minviluz Stacey University of Missouri-Columbia Shyam Barampuram University of Missouri-Columbia Nathan Hancock University of Georgia Seth Findley, University of Missouri-Columbia; Zhanyuan Zhang, University of Missouri- Columbia; Wayne Parrott, University of Georgia; Gary Stacey, University of Missouri- Columbia

Soybean is one of the world’s major commodity crops for both vegetable oil and protein. In recent years, considerable progress has been made in developing genomic resources for soybean, including the complete sequencing of the genome, which identified more than 46,300 protein encoding genes (Schmutz et al., 2010). Yet, one remaining major challenges is the elucidation of the function of these genes, especially those encoding for important agronomic traits. Insertional mutagenesis has been shown to be a powerful tool for determining gene function in several plants. Tnt1 is a retrotransposon that was originally identified in tobacco. Based on studies in a variety of plants, Tnt1 appears to be stable in mature plants but can be reactivated upon tissue culture. Our goal was to evaluate the utility of the Tnt1 retrotransposon as a mutagenesis strategy in soybean. Experiments showed that the Tnt1 element was successfully introduced into stably transformed soybean plants by A. tumefaciens- mediated transformation. Twenty-seven independent transgenic lines carrying Tnt1 insertions were generated. Southern blot analysis revealed that transposed Tnt1 copy numbers ranged from 4-19, with an average of ~8 copies per line. These insertions were stable in the progeny and showed Mendalian segregation. Analysis of 100 Tnt1 flanking sequences revealed insertions into 63 (63%) annotated genes, indicating that the element preferentially inserts into protein coding regions. Tnt1 insertions were found in all 20 soybean chromosomes, indicating that Tnt1 transposed randomly in the soybean genome. Furthermore, FISH (Fluorescence In Situ Hybridization) experiments also proved Tnt1 inserted in different chromosomes. Finally, the Tnt1 copy numbers were considerably increased upon Tnt1 reactivation experiments by using two tissue culture based approaches. Two of the Tnt1 containing lines were sufficient for the activation of the element’s transposition by tissue culture and they have been selected for future work to expand the mutant population. Thus, our data demonstrate that the Tnt1 retrotransposon is an attractive system that could be used for large-scale insertion mutagenesis in soybean.

Poster Number: 27 Genome Engineering of Soybean with Designer Nucleases

Shaun Curtin University of Minnesota Michelle Christian University of Minnesota Colby Starker University of Minnesota Justin Anderson University of Minnesota Daniel Voytas University of Minnesota Robert Stupar University of Minnesota

Site-directed gene modification remains an elusive goal for genomicists working on higher eukaryotic species like soybean. Recent advances in the field of genome engineering indicate that site-directed modifications, including targeted mutations, gene insertions and gene replacements, may soon be routine for crop species. Sequence specific nucleases that generate targeted double-stranded DNA breaks are essential for efficient site-directed genome modifications. To date, three different sequence specific nuclease systems have been used in crop plants: zinc-finger nucleases (ZFNs), TAL effector nucleases (TALENs), and LAGLIDADG homing endonucleases (also termed ‘meganucleases’). We have used genetic transformation of ZFNs to mutate single copy and duplicated soybean genes, both in hairy root and whole plant systems. Genotyping of T1 segregants identified plants that had maintained the site-directed mutation while segregating away the ZFN transgene, thus resulting in a non-transgenic mutant plant. Similar tools with increased flexibility are being developed for soybean using the TALEN approach. These promising developments may accelerate the use of precision mutagenesis and gene editing for functional genomics and trait improvement in soybean.

Poster Number: 28 Identification of Cell Specific Genes in the Soybean Root Using Laser Capture Microdissection

Jeremy Dahmen University of Missouri Gary Stacey University of Missouri

Laser capture microdissection (LCM) has been developed as a highly useful tool in analyzing cell specific transcriptomes in a wide array of organisms. Our goal is to create a transcriptome map from specific cell types in the roots of soybean. Utilizing LCM we isolated epidermis, cortex, phloem, and xylem specific cells from the soybean roots. RNA was extracted from root cell types following amplification and conversion to cDNA for RNAseq analysis. With the gene expression data obtained from RNAseq we hope to identify candidate genes specific to individual root cell types. The promoters of these cell specific transcripts will then be cloned into a GFP vector, which will then be expressed in stable transgenic soybean lines. These soybean lines will have GFP in individual root cell types, which will provide a tool allowing easy isolation of the selected root cells. This project will not only give us a comprehensive analysis of genes from in selected root cells but also provide a valuable tool for soybean researchers.

Poster Number: 29 Identification of Candidate Genes Underlying Seed Oil Concentration and Grain Yield in Soybean

Mehrzad Eskandari, Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada Elroy R. Cober, Agriculture & Agrifood Canada, Eastern Cereal and Oilseed Crop Research Centre, 960 Carling Ave, Ottawa, Ontario, K1A 0C6, Canada Istvan Rajcan, Department of Plant Agriculture, Crop Science Building, University of Guelph, 50 Stone Road East, Guelph, Ontario, N1G 2W1, Canada

Increasing oil concentration in soybean seeds has become a recent goal of soybean breeding programs due to increased demand for its use both as edible oil and feedstock for biodiesel. Sixteen putative candidate genes belonging to three important gene families (GPAT: acyl-CoA:sn-glycerol-3-phosphate acyltransferase, DGAT: acyl- CoA:diacylglycerol acyltransferase, and PDAT: phospholipid:diacylglycerol acyltransferase) have been evaluated for their involvement in triacylglycerol (TAG) biosynthesis pathways leading to oil production in soybean seeds. The sequences were compared between two cultivars with higher than average seed oil concentration, OAC Wallace and OAC Glencoe, and the published soybean sequence, Williams 82. These cultivars were used as parents to develop a population of 203 recombinant inbred lines (RIL) population, which was tested across three field environments in Ontario in 2009 and 2010. Three mutations were discovered in either the coding or non- coding regions of three DGAT isoforms between OAC Wallace and OAC Glencoe. An indel mutation within the coding region of the GmDGAT2B gene in OAC Wallace hypothesized to cause a frame shift in the gene that produces a premature stop codon was significantly associated with reduced oil concentration in RILs across the three environments. There were also two transition point mutations in the 3’ untranslated region (3’ UTR) of GmDGAT2C (A/G) and in an intron of GmDGAT1B (T/C) that were associated with grain yield at Woodstock in 2009 and Ottawa in 2010, respectively. None of the mutations showed significant effects on seed protein concentration in any of the environments. The results of this study, along with the novel candidate gene-based markers designed for GmDGAT2C and GmDGAT1B will be useful in marker-assisted selection aimed at developing soybean cultivars with increased seed oil concentration and grain yield that does not come at the expense of a reduced protein concentration.

Poster Number: 30 Allele Switching within the Soybean Aconitase-2 and Aconitase-4 loci

K. Espinosa, Dept. of Agronomy, Iowa State University R. A. Rolling, Dept. of Agronomy, Iowa State University M. Hopkins, Dept. of Biology, University of Waterloo, Ontario, Canada S. J. Lolle, Dept. of Biology, University of Waterloo, Ontario, Canada A. S. Goggi, Dept. of Agronomy, Iowa State University R. G. Palmer, Dept. of Agronomy, Iowa State University

Genetic variation was reported in several commercial cultivars of soybean [Glycine max (L.) Merr.], was traced to seed source, and was attributed to residual heterozygosity. In our studies, we have identified segregation of allelic variants at the aconitase-2 and aconitase-4 loci in sexual crosses of Minsoy (PI 27890) x Noir1 (PI 290136), and ‘BSR 101’ (PI 548519) x Minsoy, respectively. In pure line cultivars, ‘BSR 101’, and ‘Jack’ (PI 540556), we have documented multiple cases of cryptic allelic variation or “allele switching” that are stable and heritable. For both cultivars, ‘BSR 101’ and ‘Jack’, we had 64 entries, two replications, and sampled three pods, each three seeded, for a total of 1152 seed per cultivar. We have observed both single and double switches within individual 3-seeded pods. The most unusual pod originated from a homozygous aconitase-4 aa ‘BSR 101’ plant. This plant produced a pod that had the unusual aconitase-4- ab, bb for two seeds, i.e. a single switch and a double switch. The third seed in the pod was aconitase-4-aa. In ‘BSR 101’, we had 13 seed representing 10 plants with allele switches. In ‘Jack’ we had five seed, representing four plants with allele switches. The unusual ‘allele switch’ plants were allowed to self-pollinate and the resulting homozygous genotypes were used in allelism tests with the appropriate testers. All new alleles were allelic to known aconitase-2 or aconitase-4 alleles. In addition, the seeds from self-pollination of the new homozygous genotypes were true breeding for the new allele.

Poster Number: 31 Genetic Dissection of Germplasm Resources and Implications for Breeding by Design in Soybean

Junyi Gai, National Center for Soybean Improvement / MOA Key Laboratory for Biology and Genetic Improvement of Soybean / National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China

Lei. Chen, Yinghu Zhang, Tuanjie. Zhao, Guangnan Xing, Han Xing, National Center for Soybean Improvement / MOA Key Laboratory for Biology and Genetic Improvement of Soybean / National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing, China

Breeding by Design as a concept described by Peleman and van der Voort aims to bring together superior alleles for all genes of agronomic importance from potential genetic resources. This might be achievable through high-resolution allele detection based on precise QTL (quantitative trait locus/loci) mapping of potential parental resources. The present paper reviews the works at the China National Center for Soybean Improvement (NCSI) on exploration of QTL and their superior alleles of agronomic traits for genetic dissection of germplasm resources in soybeans towards practicing ‘Breeding by Design’. Among the major germplasm resources, i.e. released commercial cultivar (RC), farmers’ landrace (LD) and annual wild soybean accession (WS), the RC was recognized as the primary potential adapted parental sources, with a great number of new alleles (45.9%) having emerged and accumulated during the 90 years’ scientific breeding processes. A mapping strategy, i.e. a full model procedure (including additive (A), epistasis (AA), A x environment (E) and AA x E effects), scanning with QTLNetwork2.0 and followed by verification with other procedures, was suggested and used for the experimental data when the underlying genetic model was usually unknown. In total, 110 data sets of 81 agronomically important traits were analyzed for their QTL, with 14.5% of the data sets showing major QTL (contribution rate more than 10.0 % for each QTL), 55.5% showing a few major QTL but more small QTL (contribution rate less than 2.0 % for each QTL), and 30.0% having only small QTL. In addition to the detected QTL, the collective unmapped minor QTL sometimes accounted for more than 50% of the genetic variation in a number of traits. Integrated with linkage mapping, association mappings were conducted on germplasm populations and validated to be able to provide complete information on multiple QTL and their multiple alleles. Accordingly, the QTL and their alleles of agronomic traits for large samples of RC, LD and WS were identified and then the QTL-allele matrices were established. Based on which the parental materials can be chosen for complementary recombination among loci and alleles to make the crossing plans genetically optimized. This approach has provided a way towards breeding by design, but the accuracy will depend on the precision of the loci and allele matrices.

Poster Number: 32 Identification of Loss-of-Function Mutations Affecting a Specific R2R3 MYB Transcription Factor as the Molecular Genetic Cause for Brown Hilum and Brown Seed Coats in Glycine max.

Jason Gillman USDA-ARS Ashley Tetlow University of Missouri Jeong-Deong Lee Kyungpook National University J. Grover Shannon University of Missouri Kristin Bilyeu USDA-ARS

Modern soybean cultivars feature yellow seedcoats, with the only color variation found at the hila, although the ancestral condition is black colored seeds. Both seedcoat and hila coloration are due to phenylpropanoid pathway derivatives, principally cyanidin-3- glycoside. The genetic basis for brown hilum or seedcoats (classically, the R locus) has been utilized as a phenotypic marker for the extensive genetic maps developed for soybean, but the causative gene has not been previously identified. We utilized bulk segregant analysis to identify markers tightly linked to the R locus (brown vs. black hila) and a novel set of Simple Sequence Repeat (SSR) markers to map the causative gene to less than 200 kbp, despite the use of only 100 F6 lines. A loss of function mutation affecting a seedcoat-specific expressed R2R3 MYB transcription factor (Glyma09g36990) was identified as a strong candidate. We then investigated a broadly distributed survey of 136 soybean accessions and discerned an allelic series of loss of function mutations affecting our R locus gene candidate. Loss of function mutations were perfectly correlated with the brown hilum/seedcoat phenotype. In addition, a near perfect correlation (R2=0.96) was noted between the mRNA expression levels of the functional R gene candidate and an UDP-glucose:flavonoid 3-O-glucosyltransferase (UF3GT) gene, which encodes the catalytic enzyme responsible for the final stabilizing glycosylation of cyanidin. Our findings strongly suggest that the brown seed coat/ hilum phenotype in soybean is due to loss of function for a single seed coat-expressed R2R3 MYB transcription factor gene.

Poster Number: 33 Phosphorus Partitioning and Elemental Composition of Soybean Seeds Bearing Different Mutant Alleles of Two Soybean Seed-Specific (Glycine max (L.) Merr) ATP- binding Cassette Phytic Acid Transporter Homeologs

Jason Gillman USDA-ARS Ivan Baxter USDA-ARS Kristin Bilyeu USDA-ARS

Seed phytate is a storage repository for phosphorus and mineral micronutrients in mature soybean seeds, which presents a barrier to phosphorus and mineral bioavailability due to a lack of phytase enzyme activity in the digestive tracts of monogastric animals (e.g. humans, swine and poultry). One strategy to ameliorate these issues is the deployment of soybean varieties with reduced seed phytate. We previously identified two different recessive mutations affecting two homeologs of ATP- binding cassette phytic acid transporter (one a nonsense mutation and the other a missense mutation) which when combined are the molecular genetic cause in the EMS- induced mutant line M153. A sister low phytate EMS-induced mutant line, M766, contained a single SNP within the first intron in one transporter homeolog and a nonsense mutation in the second transporter gene. The objectives of this research were to clarify the genetics underlying the low phytate phenotype in line M766, and to determine the phenotypic effects on phosphorus partitioning in new genetic combinations of mutant alleles from M766 and M153. Genetic analysis of F2 and F3 seeds revealed that mutant alleles of both transporter genes from M766 are required for the low phytate phenotype. The transgressive inheritance of nonsense alleles affecting both transporter genes (one from M153, and one from M766) led to the production of viable seeds that contained transgressive reductions in total seed phytate and significantly higher levels of inorganic phosphate than has been reported for non- transgenic soybean material. This new information will allow the efficient production and molecular selection of soybeans with even greater reductions of phytate for further improved quality soybean meal.

Poster Number: 34 Identification of Genes/Loci and Functional Markers for Seed Oil Quality Improvement by Exploring Soybean Genetic Diversity

Wolfgang Goettel, Plant Genetics Research Unit, USDA-ARS, Donald Danforth Plant Science Center, St. Louis, Missouri 63131 Pengyin Chen,115 Plant Science Building, University of Arkansas, Fayetteville, Arkansas 72701 Greg Upchurch, Soybean & Nitrogen Fixation Unit, USDA-ARS, Raleigh, NC 27696 Yong-Qiang Charles An, Plant Genetics Research Unit, USDA-ARS, Donald Danforth Plant Science Center, St. Louis, Missouri 63131

The difference in seed oil composition and content among soybean genotypes can be attributed mostly to variations in transcript sequences and/or transcript accumulation of oil-related genes expressed in seeds. We applied the Illumina HiSeq 2000 system to sequence RNA populations in soybean seeds from nine genotypes varying in profiles of lipid species and/or total oil content. For each genotype, an average of 35 million 100 bp paired-end reads were generated, and 30,167 annotated transcripts from 28,270 genes were detected in seeds at mid-maturation stage. A wide range of bioinformatic algorithms were used to determine the variation in transcript accumulation, transcript splicing, and transcript isoforms among the genotypes. Putative SNPs in those transcripts and indels in genes and genome segments were also identified. M23 is a previously characterized X-ray mutant line with a mid-oleic acid phenotype and has a deletion of a 160 kb genome segment encoding a delta-twelve fatty acid desaturase and 19 other proteins. We detected no or dramatically lower sequence reads aligning to those genes in M23 compared to the other genotypes, which validated the effectiveness of our approach. Pros and cons of this functional genomic approach to discover genes, gene variations and functional markers for seed oil quality improvement will be discussed in detail. Polymorphisms discovered by this project could potentially be developed into a set of functional markers for breeders to design effective crossing and marker assisted selection strategies for oil quality improvement.

The research is supported by the United Soybean Board and USDA-ARS.

Poster Number: 35 SoyBase: The USDA-ARS Soybean Genetics and Genomics Database

David Grant USDA-ARS-CICGRU and Iowa State University, Ames, Iowa Rex Nelson USDA-ARS-CICGRU, Ames, Iowa Kevin Feeley USDA-ARS-CICGRU, Ames, Iowa Robert Baker USDA-ARS-CICGRU, Ames, Iowa Nathan Weeks USDA-ARS-CICGRU, Ames, Iowa Steven Cannon USDA-ARS-CICGRU and Iowa State University, Ames, Iowa Randy Shoemaker USDA-ARS-CICGRU and Iowa State University, Ames, Iowa

SoyBase, the USDA-ARS soybean database, is a central location for genetics, genomics and related data. The main SoyBase page (http://soybase.org) contains a News section, links to upcoming conferences and meetings, and acts as a portal to the Soybean Breeder’s Toolbox (SBT). The SBT is the user interface to the data in SoyBase and is constantly being upgraded and improved with new genetic and genomic data and bioinformatic tools. The SBT is organized around two related data types: the composite genetic and physical maps and the whole genome sequence. Genetic and physical maps allow side-by-side displays of multiple maps for comparing homoeologous regions of the genome. In addition to RFLP and SSR markers, QTL and the final FPC-based Williams 82 physical map, SNP-based markers are included in the SBT. The sequence-based genetic markers allow detailed integration of the genetic and sequence maps. Users can choose to see sequenced genetic markers, soybean BACs, repetitive sequences, EST-based unigene sets, RNA expression, and JGI automated gene calls along with many other data types in the context of the Williams 82 genomic sequence in the SoyBase Genome Browser. Features on the genetic, physical and sequence maps (eg. genes, markers, QTL, BACs and FPC contigs) are linked to additional detailed textual data in the SBT, and also provide connections between the genetic maps and the whole genome sequence. Controlled vocabularies for soybean growth, development and phenotypic traits have been developed and integrated with the Plant Ontology Consortium and Plant Trait Ontologies. Soybean ontologies are linked as appropriate to genes and QTL in the SBT, thus allowing searching by trait or developmental stage as well as by gene or QTL name or annotation.

Poster Number: 36 Soybean Seed Composition Improvement through Molecular Breeding

Katherine Hagely University of Missouri Kristin Bilyeu

Nearly 75% of soymeal produced is utilized in animal feeds for poultry, swine, and pet food. Soymeal contains many anti-nutritional factors that sequester metabolic energy and prevent efficient weight gain. Lectin binds amino acids in animal guts and prevents their uptake. Raffinose family oligosaccharides (RFOs) are not digestible by monogastric animals due to their lack of alpha-galactosidase enzyme activity. Investigation into the molecular genetic basis of soybean lines with an ultra-low RFO phenotype has enabled the development of molecular marker assays for this ultra-low RFO and low lectin traits. These assays are currently being used by plant breeders in both the public and private sectors to breed soybeans that contain higher metabolizable energy and for use in animal feeds.

Poster Number: 37 The comparison Of Malondealdehyde, Total Antioxidant and Isoflavonoides, Levels in Soymilk and Textured Soy Protein

Parichehr Hanachi Alzahra University Latifah Abdul Latif

Soya does not constitute a part of the general diet in Iran, Isoflavones were observed to have an antioxidant activity in vitro and in vivo, study. The purpose of this study were comparison of isoflavonoides, malondialdehyde (MDA), total antioxidant capacity (TAC) in soymilk and textured soy protein (TSP). This study were determined isoflavonoides such as daidzein and genistein of TSP and soymilk, by HPLC system, for assay MDA, it was conjugated with thiobarbituric acid; the absorbance at 535 nm was read against blank. TAC was measured by the use of ferric reducing antioxidant power (FRAP) method. Statistical analysis was performed using SPSS ver,16.0 for Windows. The level of P < 0.05 were considered statistically significant. The concentration of genistein in soymilk and TSA, were higher than daidzein, values, however, MDA level was significantly (p<0.05) less and TAC level was significantly more ( p<0.05) in soymilk compared with TSA. The results obtain in this study can serve as basis for estimating isoflavones and antioxidant amount of TSA and soymilk can be consumed by individuals as related to main isoflavones content.

Keywords: Antioxidant, Isoflavonoides, malondialdehyde, soymilk

Poster Number: 38 Methodology to Identify Unique Genetic Sources of Asian Soybean Rust Resistance from the USDA Germplasm Collection

Donna Harris University of Georgia David L. Hyten, Perry B. Cregan, James W. Buck, Zenglu Li, and H. Roger Boerma

Eighty-five crosses between a susceptible (S) elite cultivar or breeding line and Asian soybean rust (SBR) resistant (R) plant introduction have been made in the summers of 2006 through 2011. These plant introductions have previously shown a high level of SBR resistance across multiple years and locations. One-hundred and twenty individual F2 plants along with the parents are grown in the greenhouse in Griffin, GA for phenotyping purposes. Plants are inoculated using spores from greenhouse stock soybean plants that originally were inoculated with spores collected from field-grown soybean plants as well as kudzu plants around Georgia during the summer. DNA is extracted and bulked together in equal concentrations to make a susceptible and resistant bulk sample for bulk segregant analysis (BSA). The bulked DNA samples along with their parents are genotyped using the 1536 SNP markers on the GoldenGate platform (Illumina, San Diego, CA). GenCall software (Illumina, San Diego, CA) is used to identify candidate regions of interest where the susceptible bulks have the same alleles as the susceptible parent in the GenCall output. Results from the GenCall output determine if the resistance gene in the resistant parent maps to a previously identified region of the genome harboring Rpp1, 2, 3, 4, 5, or 6 or a different region of the soybean genome. If BSA identifies a putative unique resistance gene from a plant introduction, 140 F2:3 lines and their parents are phenotyped in the greenhouse. The greenhouse experiments are conducted using 12 plants per individual F2:3 line and 48 plants of each parent randomly dispersed through the experiment. SNP markers from the genomic regions identified by BSA are converted to the KASP assay (KBioscience, Beverly, MA) to genotype the entire population and locate the QTL positions.

Poster Number: 39 Characterization of Promoter Sequences from Soybean Pathogen-Responsive Genes

Yanyu He University of Illinois Urbana-Champaign Anne Dorrance OARDC/The Ohio State University Paul Rushton South Dakota State University John Finer OARDC/The Ohio State University Steven J. Clough, USDA-ARS, University of Illinois Urbana-Champaign

This research is designed to identify and characterize soybean genes and promoter sequences that respond to pathogens like Sclerotinia, Fusarium virguliforme, and Psuedomonas syringae. qRT-PCR and microarray analyses on inoculated soybean foliar tissue are being conducted to identify early- and late-induced genes and a wide range of their associated promoters. We have selected 11 pathogen responsive candidate genes based on in-house microarray studies and published papers. Fusion of GFP to promoters of these genes is underway in collaboration in the Finer lab at The Ohio State University for analysis of function. The Rushton lab is using computational analysis of promoter sequences to identify common elements within pathogen responsive promoters. Additionally, we obtained full-length cDNA of select pathogen- responsive genes of interest and are cloning them into Agrobacterium overexpression vectors for Arabidopsis and soybean transformation. These transgenic plants will be assayed for altered responses to pathogens. This ongoing research project will increase our understanding of promoter function, identify genes fairly specific to pathogen infection, lead to the development of markers that are associated with pathogen resistance, and allow us to generate a collection of soybean promoters for basic research and possible transgenics with enhanced resistance.

Poster Number: 40 Novel Promoter Elements Associated with Wound Responses in Soybean

Carlos Hernandez-Garcia The Ohio State University/OARDC Michelle Jones The Ohio State University/OARDC Paul Rushton South Dakota State University John Finer The Ohio State University/OARDC

Promoters are the main regulators of gene expression at the transcriptional level. Promoter functionality is dictated by the promoter elements generally located within the promoter regions. In soybean, only a few promoters have been well studied, and soybean wound-inducible promoters and their contributing regulatory elements have received little to no attention. As plants show similar responses to mechanical wounding, pathogen invasion and damage from chewing insects, studies of wound- inducible promoters and their elements could provide insights to the general mechanisms of regulation of stress-responsive genes. Here we studied induction of ten GmERF (Glycine max Ethylene Response Factor) genes and their promoters, and validated the elements identified within the promoter sequences. RT-PCR analysis of GmERF transcripts showed high level accumulations in soybean seedlings following wounding or treatment with either methyl jasmonate or ethylene. Four GmERF promoters were subsequently isolated, fused with GFP, and reintroduced into soybean for expression analysis. In transgenic plants, the re-introduced GmERF promoters directed low basal GFP expression in roots, pods, the epidermis, and vascular tissues. However, these promoters were highly inducible by wounding in cotyledons, hypocotyls and leaves. Wound-inducible expression was not detected in wounded roots, indicating that wound induction of these GmERF promoters is organ-dependant. Deletion analysis and promoter element identification for the GmERF3 promoter suggests an intricate regulation of expression. Candidate regulatory elements from specific promoter regions and representing previously identified components as well as novel sequences were isolated and characterized. Promoter elements likely responsible for induction of gene expression after mechanical wounding were validated using transient expression analysis and site-directed mutagenesis. This study will increase our understanding of ERF promoter functionality and expand the toolbox of soybean wound-inducible promoters and promoter elements for potential use in both basic and applied research.

Poster Number: 41 Characterization of A Soybean Lanceolate Leaf Mutation Induced by an Activation Tag Harboring T-DNA Insertion

Khang Hoang Center for Plant science innovation, University of Nebraska-Lincoln Hanh Nguyen Center for Plant science innovation, University of Nebraska-Lincoln Shirley Sato, Manmeet Singh, Saadia Bihmidine, and Thomas Clemente

Within a soybean population containing T-DNA insertions which harbor a Ds-transposon delineated activation tag element, an event was observed with an abnormal morphological characteristics. The event, designated 850-15, displayed a phenotype consisting of grey trichomes, yellow pods, increased plant height, white hilum, and lanceolate leaflets under greenhouse conditions, which faithfully traced with the inheritance of the T-DNA. Junction fragment analysis revealed that T-DNA carrying activation tag landed into an exon of 18S ribosomal RNA gene, however, due to sequence redundancy of the junction fragment within the genome we could not easily assign a chromosome location for the T-DNA element. To determine if the phenotype is triggered by either an insertion mutation, or the result of the activation tag, which theoretically might activate/or down-regulate other genes in the locus, we are stacking the 850-15 event with the Ac-transposase in order to mobilize the Ds-activation tag out of the locus. In addition, we are creating gene stacks with 850-15 with transgenes designed to enhance photosynthetic capacity. Thereby creating a soybean with altered leaf morphology to permit superior light penetrance in the canopy, coupled with net photosynthesis improvement, with the long term goal of phenotyping populations derived from the stack under field conditions for agronomic and quality parameters.

Poster Number: 42 The Role of Phytohormones in Soybean Aphid Suppression of Plant Defenses

Jessica Hohenstein Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University Gustavo MacIntosh Department of Biochemistry, Biophysics, and Molecular Biology, Iowa State University

Soybean aphids are phloem-feeding insects that can cause significant economic losses. Recent transcriptome analyses have identified several phytohormone pathways, including some normally associated with abiotic stress, that are activated in soybean in response to aphid colonization. Gene expression and metabolomics studies suggest that aphids are able to block effective plant defense responses, which may not only favor aphid colonization but also may affect other pests that attack soybean during multiple-pest interactions. The mechanism employed by aphids to suppress plant defenses and successfully colonize susceptible soybeans is currently not known.

We are investigating the role that phytohormones play during the progression of aphid infestation; specifically how abiotic stress signals such as abscisic acid may be induced by aphids, resulting in a decoy response that suppresses effective defenses. In growth chamber experiments, several ABA-responsive genes including a C2H2-type zinc finger protein, SCOF-1, were highly induced in the leaves of aphid-infested plants. SCOF-1 is a transcriptional activator that enhances ABRE-dependent gene expression, thus controlling ABA responses. Using a transgenic hairy root system, we knocked down SCOF-1 and found that aphids performed significantly lower on the scof-1-deficient roots than on control roots.

This apparent use of ABA to suppress effective defenses may have implications in how multiple pests interact through the same plant. Previous results from our laboratory have shown that aphid colonization can increase the severity of soybean cyst nematode infestations. We performed an analysis of gene expression in leaves and roots of aphid- infested plants and found significant systemic signaling to the roots in these plants. However, the phytohormone signals activated in roots are different than those activated in leaves.

Poster Number: 43 A Genome-Wide Association Study to Identify Loci Associated with Seed Protein Concentration in Soybean

Eun-Young Hwang Plant Science and Landscape Architecture, Univ. of Maryland, College Park, Maryland 20742 Qijian Song Soybean Genomics and Improvement Lab, USDA, ARS, Beltsville, Maryland 20705 Gaofeng Jia Soybean Genomics and Improvement Lab, USDA, ARS, Beltsville, Maryland 20705 James Specht Agronomy & Horticulture, University of Nebraska, Lincoln, Nebraska 68583 Jose Costa, Plant Science and Landscape Architecture, Univ. of Maryland, College Park, Maryland 20742; Perry Cregan, Soybean Genomics and Improvement Lab, USDA, ARS, Beltsville, Maryland 20705

Genome–Wide Association Studies (GWAS) are applied to detect the location of qualitative genes and quantitative trait loci using the correlation between DNA marker alleles or haplotypes and the phenotypic expression of a trait of interest. In this research, GWAS was performed to detect loci controlling seed protein concentration in soybean. A total of 42,368 SNPs were analyzed in a diverse set of Glycine max (L.) Merr. germplasm accessions from the USDA Soybean Germplasm Collection. Field tests were conducted to evaluate seed protein concentration of the accessions using a randomized complete block design with four replicates at Beltsville, MD and two replicates at Lincoln, NE. Total seed nitrogen was determined using a LECO CHM 2000 analyzer (LECO, St. Joseph, MI) in order to obtain an estimate of seed protein concentration. Analysis of variance was conducted and population structure of the 302 accessions was estimated by the Admixture program v. 1.22 (Alexander et al. 2009). Association analysis was performed with the mixed linear model approach implemented by TASSEL (Bradbury et al. 2007). GWAS detected 17 loci on 10 chromosomes showing significant association (-logP>3) with seed protein concentration including associations at the 27-30 Mbp region on Gm20, at 14.7 Mbp on Gm06, and at 8.9 Mbp on Gm17. Of the 17 associations, 12 corresponded with the location of previously reported seed protein QTL. This result indicates that GWAS was successful in identifying loci controlling seed protein concentration in soybean germplasm accessions.

Poster Number: 44 QTL Mapping and Primary Screening of Candidate Genes for Shoot Ureide and Nitrogen Concentration in a Soybean (Glycine max)

Sadal Hwang University of Arkansas C. Andy King Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72704 Marilynn K. Davies Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72704 Jeffery D. Ray Crop Genetics and Production Research Unit, USDA-ARS, Stoneville, MS 38776 Perry B. Cregan Soybean Genomics and Improvement Laboratory, USDA-ARS, BARC-West, Beltsville, MD Larry C. Purcell Department of Crop, Soil, and Environmental Sciences, University of Arkansas, Fayetteville, AR 72704

Many studies have reported that N2 fixation in soybean (Glycine max) is more sensitive to drought than leaf gas exchange and that shoot ureide and N concentrations were highly associated with the sensitivity of N2 fixation to drought. Genotypic differences in shoot ureide and N concentration were evaluated using a high density linkage map (< 20 cM) constructed from an F5-derived population of KS4895 x Jackson with 97 RILs. Shoot ureide and N were measured from R4 to early R5 under irrigated conditions in 3- year field trials. Heritability for shoot ureide and N was 0.73 and 0.60, respectively, and these traits were closely associated (r = 0.72) with each other. For shoot ureide, 5 QTLs were identified using composite interval mapping (CIM) on LGs, C2 (1), K (1), F (1), and L (2) with R2 values ranging from 0.11 to 0.31 and additive effects ranging from 2.32 to 5.98 uM/g. In multiple interval mapping (MIM), 2 QTLs were coincidently positioned on LGs, F and L. For shoot N, 4 QTLs were detected using CIM on LGs, F (3) and J (1) with R2 values ranging from 0.11 to 0.25 and additive effects ranging from 0.085 to 0.156 %. In MIM, one QTL had the same position on LG F as that identified with CIM. All QTLs except for one on LG L would be stable over the years considering QTL x Year interaction results from CIM and MIM. The QTL on LG F for shoot ureide and N appeared to be pleiotropic considering their overlapping LOD intervals and high correlation. Identified QTLs for shoot ureide and N were related with traits which were involved in plant growth, seed or yield components and biotic or abiotic stress based on previous QTL information provided by SoyBase (http://www.soybase.org). A GO analysis (http://www.geneontology.org) was initially used to search for metabolic process-related genes in QTL regions on LGs, F and L in which QTLs were identified with both CIM and MIM analyses. Two notable predicted genes from the Glyma 1.0 gene set were found for shoot N (carbonic anhydrase, Glyma13g39920) and ureide (inosine-uridine nucleoside hydrolase, Glyma19g37830) on LGs, F and L, respectively. We expect that QTL information for these traits could be also used to select lines which are tolerant with N2 fixing ability under water-deficit.

Poster Number: 45 Characterization of a Fast Neutron-Induced Soybean Mutant Using Next-Generation Sequencing

Woon Joo Hwang Seoul National University Kwang-Soo Han Seoul National University Yang Jae Kang Seoul National University Sang Rae Shim Seoul National University Moon Young Kim, Suk-Ha Lee, Seoul National University

Mutagenesis approach in combination with whole genome sequencing has become an important role in genetic and molecular biology study and breeding of crop plants. In this study, we screened the fast neutron M4 10,000 soybean mutant plants based on morphological phenotypes of agronomically important traits and characterized the mutant of interest using resequencing. Screened mutants showed abnormal phenotypes in plant heights, seed sizes, color of leaves, number of leaves, maturity and number of branches etc. Among them, the mutant displaying short plant height and bush type of growth habit was selected to perform resequencing for identification of the altered genomic regions. Mutant sequence reads generated by paired-end shotgun library were mapped on a draft soybean reference soybean (Glycine max cv. Williams 82). The paired-end DNA sequences of 21.6 Gb produced by Illumina Hi-seq produced 21 fold sequence depth. Among the predicted deletion sites, a total of 3 deletion regions were confirmed by PCR. Glyma03g02390 gene and Glyma03g03560 gene were involved in the deletion regions. Glyma03g02390 gene was related to AMP binding, catalytic activity, cofactor binding and metabolic process of cell growth and Glyma03g03560 gene was concerned to oxygen binding, defense response to bacterium, and especially process of indole acetic acid (IAA) biosynthesis. These genes detected in this mutant will be studied their molecular function in stunted phenotype.

Poster Number: 46 Utilizing Next Generation Sequencing in Soybean Product Development

David Hyten DuPont Pioneer John Woodward DuPont Pioneer Les Kuhlman DuPont Pioneer Matt King DuPont Pioneer Yun Zhang, DuPont Pioneer; Bryce Daines, DuPont Pioneer; Scott Sebastian, DuPont Pioneer; Stephane Deschamps, DuPont; Josh Shendelman, DuPont Pioneer; Edwin Mendez, DuPont Pioneer; Andrea Kalvig, DuPont Pioneer; Kevin Hayes, DuPont Pioneer

DuPont Pioneer has had tremendous success in utilizing molecular markers throughout the soybean product development breeding scheme to increase productivity through the Accelerated Yield Technology (AYTTM) system and the utilization of native traits. Next Generation sequencing has created a revolution in the world of genomics that is enabling DuPont Pioneer to look past the use of molecular markers to a fuller integration of genomics within product development. Through the use of this technology we have resequenced 183 soybean lines which include the lines that make up soybean’s ancestral base, several key lines throughout DuPont Pioneer’s history and many elite lines which are being used as parents in our current product development. This resequencing dataset has enabled us to discover a large proportion of the variation that is present within our germplasm for soybean improvement and allows us to identify regions of the genome where diversity has become extremely low. This data is also being used to increase our ability to identify and delimit regions of the genome that contribute to important native traits enabling discovery and better marker development. In addition, this resequencing data allows an unprecidented view into the inheritance of progeny lines within breeding populations. Genomics technology such as next generation sequencing has allowed a further integration of the use of genomics into the product development strategy which will result in higher performing products within farmer’s specific growing environments.

Poster Number: 47 Marker-Assisted Backcrossing to Improve Fatty Acids in Soybean

Abby E. Isabelle National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211 Tri D. Vuong National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211 J. Grover Shannon National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211 Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211

Increased oleic acid and decreased linolenic acid in soybeans enhance oxidative stability and human nutrition. The marker-assisted backcrossing (MABC) project aims to incorporate mutant alleles conferring oil quality traits into elite commercial germplasm. Using the Roche LightCycler SimpleProbe genotyping platform, we have tested and optimized single nucleotide polymorphism (SNP) assays for mutant alleles in multiple sources, including fad2-1a and fad2-1b alleles for high oleic acid, and fad3a, fad3b, and fad3c alleles for low linolenic acid. Additionally, we optimized a gel electrophoresis and melting curve protocol to detect the low linolenic source of the deleted fad3a gene and a novel fad3a mutation from a PI 361088B, respectively. To develop new soybean varieties with high oleic and low linolenic levels that are stable in northern environmental conditions, over 500 F2 plants derived from different crosses of mutant accessions were genotyped and utilized for the MABC program. New crosses were produced with several elite lines. As part of the United Soybean Board project for improved oil functionally, we provide genotyping support on soybean lines provided by Northern soybean breeders for their marker-assisted selection breeding programs. Our future goals are to continue MABC for the Northern soybean breeding programs and to discover new genes/alleles from novel sources of fatty acids in an effort to develop new soybean varieties with elevated oleic acid and reduced linolenic acid.

Poster Number: 48 pGmUTE; A Novel, Efficient, and Highly Effective Gene Silencing System for Soybean

Thomas Jacobs University of Georgia Peter R. LaFayette, Lila O. Vodkin and Wayne A. Parrott

Most gene-silencing attempts involve the use of hairpin constructs, in which a section from the target gene is cloned as an inverted repeat separated by an intron or a spacer sequence. An alternative approach is to utilize the trans-acting siRNA (tasiRNA) pathway. Accordingly a 22-nt miRNA recognition site is fused to a section of a target gene. The result is the production of siRNAs specific to the target gene. Small RNA sequence analysis from hairy root cultures was used to identify several candidate miRNAs that may induce the tasiRNA pathway in soybean, one of which is miR1514. Next, three genes, NFR, P450, which are native, and a GFP transgene were targeted by fusing the recognition site for miR1514 to an approximately 300-nt long section of each target gene. RT-PCR analysis confirmed the silencing of all targeted genes. Sequencing of RNA from hairy root events confirmed the production of small RNAs specific to targeted sequences 3’ from the miR1514 recognition site. Loss of phenotype associated with silencing is easily observed when GFP is targeted. Together, these results indicate that miR1514 is a bona fide tasiRNA-inducing miRNA in soybean and can be useful to induce gene silencing. Such an approach is simpler than the construction of hairpin vectors. The approach used here to identify and use tasiRNA- inducing miRNA loci should be applicable to other crops as well.

Poster Number: 49 Dynamic Genetic Features of Chromosomes Revealed by Comparison of Soybean Genetic and Sequence-Based Physical Maps

Soon-Chun Jeong Korea Research Institute of Bioscience and Biotechnology Woo Kyu Lee, Namshin Kim, Jiwoong Kim, Jung-Kyung Moon, Namhee Jeong, Ik- Young Choi, Sang Cheol Kim, Won-Hyong Chung, Hong Sig Kim, Suk-Ha Lee, Soon- Chun Jeong

Despite the intensive soybean [Glycine max (L.) Merrill] genome studies, the high chromosome number (20) of the soybean plant relative to other major crops has hindered the development of a high-resolution genomewide genetic map derived from a single population. Here, we report such a map, which was constructed in an F15 population derived from a cross between G. max and G. soja lines using indel polymorphisms detected via a G. soja genome resequencing. By targeting novel indel markers to marker-poor regions, all between marker distances were reduced to under 6 cM on a genome scale. Comparison of the Williams 82 soybean reference genome sequence and our genetic map indicated that marker orders of 26 regions were discrepant with each other. In addition, our comparison showed 7 misplaced and 2 absent markers in the current Williams 82 assembly and 6 markers placed on the scaffolds that were not incorporated into the pseudomolecules. Then, we showed that, by determining the missing sequences located at the presumed beginning points of the 5 major discordant segments, these observed discordant regions are mostly errors in the Williams 82 assembly. Distributions of the recombination rates along the chromosomes were similar to those of other organisms. Genotyping of indel markers and genome resequencing of the two parental lines suggested that some marker-poor chromosomal regions may represent introgression regions, which appear to be prevalent in soybean. Given the even and dense distribution of markers, our genetic map can serve as a bridge between genomics research and breeding programs.

Poster Number: 50 QTL Identification and Correlation Analysis of Seed Oil, Protein Content and Yield in Soybean

Guo-Liang Jiang South Dakota State Univewrsity Xianzhi Wang South Dakota State University Marci Green South Dakota State University Roy Scott USDA-ARS David Hyten, USDA-ARS; Perry Cregan, USDA-ARS

Soybean seeds contain high levels of protein and oil useful for human consumption. Increasing oil and protein content as well as yield are the main objectives of soybean breeding. However, previous studies show that there are negative correlations among seed oil, protein concentration and yield, making it difficult to improve the traits simultaneously. The objectives of this study were to identify QTLs for oil, protein content and yield in soybean, to evaluate the genetic effects of individual QTLs and QTL combinations, and to explore the relationships among these traits using marker-based linkage information. A population of recombinant inbred lines (RILs) derived from the cross of SD02-4-59 x A02-381100 was planted in a RCBD and evaluated for seed yield, oil and protein content in five environments over three years. A soybean genetic linkage map constructed with single nucleotide polymorphism (SNP) markers and simple sequence repeat (SSR) markers was used to conduct QTL mapping. By using composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and/or the interval mapping (IM) method, a total of 24 QTLs for the three traits were repeatedly detected in at least two environments. The QTLs on linkage group D2 (Chromosome 17) and I (Chromosome 20) for oil, protein content and yield, the QTL on linkage group E (Chromosome 15) for oil and protein content, and the QTL on linkage group O (Chromosome 10) for oil content and yield were situated in close proximity to one another (peak markers linked <5cM). This suggests that these QTLs might have pleiotropic effects. This molecular information may provide a better understanding of the relationships among oil, protein content and yield, and may also be useful for breeding higher-yielding and better quality soybean varieties. Comparisons of multiple- locus combinations indicated that cumulative effects of QTLs were significant for all traits. QTL pyramiding by molecular marker-assisted breeding would be an appropriate strategy for improvement of oil, protein content and yield in soybean.

Poster Number: 51 GRP7, A Substrate of Pseudomonas syringae Type III Effector HopU1, Plays a Role in Plant Innate Immunity

Anna Joe School of Biological Science, Center for Plant Science Innovation, University of Nebraska, Lincoln, Nebraska 68588 USA Shirley Sato Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68588 USA Hanh Nguyen Center for Biotechnology, University of Nebraska, Lincoln, Nebraska 68588 USA Tom Clemente Center for Plant Science Innovation, Center for Biotechnology, Department of Agronomy and Horticulture, University of Nebraska, Lincoln, Nebraska 68588 USA James R. Alfano Center for Plant Science Innovation, Department of Plant Pathology, University of Nebraska, Lincoln, Nebraska 68588 USA

The bacterial pathogen Pseudomonas syringae uses a type III secretion system to inject type III effectors into plant cells and suppress plant immunity. The P. syringae pv. tomato DC3000 type 3 efferctor HopU1 was determined to be a mono-ADP- ribosyltransferase that can use several RNA-binding proteins as substrates. One of these proteins, GRP7 was shown to be involved in innate immunity and Arabidopsis mutants lacking GRP7 were more susceptible to P. syringae. HopU1 ADP-ribosylates an arginine residue in position 49 of GRP7, which is within its RNA-recognition motifs. We found that ADP-ribosylated GRP7 was reduced in its ability to bind RNA. Complementation of the grp7 mutant with wild type GRP7 but not the GRP7R49K mutant restored normal immune response. This indicates the amino acid that is the site of ADP-ribosylation is critical for GRP7's function. Recently, we found that plants over- expressing GRP7 were more resistant to P. syringae and other pathogens demonstrating that GRP7 plays a broadly important role in innate immunity. To investigate if this resistance could be translated into crop plants we identified 4 GRP7 homologs in soybean. Two of these genes, Glyma06g01470 and Glyma11g12480, that are highly similar to Arabidopsis GRP7, 78 % and 82 % identical respectively, were chosen for further study. We sub-cloned these Gmgrp7s and Atgrp7 into the binary vector pPTN200 with 35S promotor and terminator and transformed them into soybean using the Agrobacterium-mediated method. The immune phenotypes of these soybean transgenics will be presented.

Poster Number: 52 Using RNASeq to Profile Soybean Seed Development from Fertilization to Maturity

Sarah I. Jones University of Illinois Lila O. Vodkin

To understand gene expression networks leading to functional properties and compositional traits of the soybean seed, we have undertaken a detailed examination of soybean seed development from a few days post-fertilization to the mature seed using Illumina high-throughput transcriptome sequencing (RNASeq). RNA was sequenced from seven different stages of seed development, yielding between 12 million and 76 million sequenced transcripts. These have been aligned to the 79,000 gene models predicted from the soybean genome recently sequenced by the Department of Energy Joint Genome Institute. Data are given in RPKMs, representing reads per kilobase per million mapped reads. By taking into account both the total number of mapped reads per sample and the length of the gene model, RPKMs can be used to compare across different gene models and samples. Genes involved with some storage proteins such as glycinin and beta-conglycinin had their highest expression levels at the stages of largest fresh weight, confirming previous knowledge that these storage products are being rapidly accumulated before the seed begins the desiccation process. Over one hundred gene models were identified with high expression exclusively in young seed stages, starting at just four days after fertilization. These were annotated as being related to many basic components and processes such as histones, proline-rich proteins, chlorophyll-binding proteins, membrane-related proteins, and proteins involved in ubiquitination. The high expression of these genes may reflect the rapid expansion of cells and tissues during these early stages of seed development. There are also a large number of gene models annotated as senescence-associated proteins with high expression at these early seed stages, suggesting the need for further study of their functions in tissues other than senescing seeds and leaves. Other gene models, including those annotated as transcription factors and seed proteins, showed high expression in the dry, mature seeds, perhaps indicating the preparation of pathways needed later, in the early stages of imbibition. Many highly-expressed gene models at the dry seed stage are, as expected, annotated as hydrophilic proteins associated with low water conditions, such as late embryogenesis abundant (LEA) proteins and dehydrins, which help preserve the cellular structures and nutrients within the seed during desiccation. Many gene models with unknown annotations showed high expression at both very young and dry, mature stages, suggesting intriguing areas for future research. This work was supported by the USDA, Illinois-Missouri Biotechnology Alliance, United Soybean Board, and the Illinois Soybean Association.

Poster Number: 53 Bridging the Gap Between Soybean Translational Genomics and Breeding With Soybean Knowledge Base (SoyKB)

Trupti Joshi University of Missouri - Columbia Michael R. Fitzpatrick University of Missouri - Columbia Levi D. Franklin University of Missouri - Columbia Shiyuan Chen University of Missouri - Columbia Jianlin Cheng, University of Missouri - Columbia; Gary Stacey, University of Missouri - Columbia; Henry Nguyen, University of Missouri - Columbia; Dong Xu, University of Missouri - Columbia

Many genome-scale data are available in soybean including genomic sequence, transcriptomics (microarray, RNA-seq), proteomics and metabolomics datasets, together with growing knowledge of soybean in gene, microRNAs, pathways, and phenotypes. This represents rich and resourceful information which can provide valuable insights, if mined in an innovative and integrative manner and thus, the need for informatics resources to achieve that.

Towards this we have developed Soybean Knowledge Base (SoyKB), a comprehensive all-inclusive web resource for soybean translational genomics and breeding. SoyKB handles the management and integration of soybean genomics and multi-omics data along with gene function annotations, biological pathway and trait information. It has many useful tools including Affymetrix probeID search, gene family search, multiple gene/metabolite analysis, motif analysis tool, protein 3D structure viewer and download/upload capacity for experimental data and annotations. It has a user-friendly web interface together with genome browser and pathway viewer, which display data in an intuitive manner to the soybean researchers, breeders and consumers.

SoyKB has new innovative tools for soybean breeding including a graphical chromosome visualizer targeted towards ease of navigation for breeders. It integrates QTLs, traits, germplasm information along with genomic variation data such as single nucleotide polymorphisms (SNPs) and genome-wide association studies (GWAS) data from multiple genotypes, cultivars and G.soja. QTLs for multiple traits can be queried and visualized in the chromosome visualizer simultaneously and overlaid on top of the genes and other molecular markers as well as multi-omics experimental data for meaningful inferences.

SoyKB can be publicly accessed at http://soykb.org

Poster Number: 54 Glycerol-3-phosphate, An Inducer of Defense against Diverse Pathogens in Soybean

Aardra Kachroo University of Kentucky Qing-ming Gao University of Kentucky Da-Qi Fu University of Kentucky Kentaro Sekine University of Kentucky Devarshi Selote, Pradeep Kachroo

The health and disease physiologies of most organisms are intricately linked and primary metabolic components often regulate disease signaling. For example, energy- generating pathways, such as the pentose phosphate, glycolytic, and fatty acid metabolic pathways, participate in the defense physiologies of plants and animals alike. Glycerol-3-phosphate (G3P), an obligatory component of energy-producing reactions including glycolysis and glycerolipid biosynthesis, constitutes a case in point. Many human disorders are associated with alterations in G3P levels and the activities of G3P- metabolizing enzymes. Plants are no different; G3P and its metabolism are intricately linked to the plants’ ability to defend itself against a variety of pathogens. We have shown that G3P is important for plant defense to fungal pathogens; genetic mutations affecting G3P synthesis in the ‘model’ plant Arabidopsis thaliana enhance susceptibility to hemibiotrophic and necrotrophic fungi. On the other hand, plants accumulating increased G3P show enhanced resistance to these pathogens. G3P also promotes defense against other necrotrophic fungi and bacterial pathogens. This defense related role of G3P is conserved amongst diverse plants because soybean plants defective in G3P biosynthesis show enhanced susceptibility to the oomycete Phytophthora sojae. For example, soybean plants silenced for two related isoforms of the G3P dehydrogenase (G3Pdh) genes are compromised in defense against P. sojae. Likewise, soybean plants silenced for the glycerol kinase (GK) gene also show enhanced susceptibility to P. sojae. Recently, we also showed that G3P is an essential mobile regulator of systemic acquired resistance (SAR) in Arabidopsis and soybean alike. Exogenous G3P induced SAR in soybean, and soybean plants silenced for the G3Pdh or GK genes are impaired in SAR. SAR is a highly desirable form of induced resistance because it provides long lasting and broad-spectrum resistance in plants. However, SAR induction is often associated with the accumulation of the phytohormone salicylic acid (SA), which can induce highly energy intensive processes and affect plant growth and development. Notably, G3P-induced SAR does not involve the induction of SA. This avoids the diversion of carbon, nitrogen, and energy away from the plants’ primary metabolic pathways, which can negatively impact growth and development. Consequently, G3P-induced immunity has direct practical implications for crop improvement without negatively impacting yield.

Poster Number: 55 Genetic Analysis and Mapping QTLs with Epistatic Effects for Seed Coat Cracking In Soybeans

Sungtaeg Kang Dankook University Bo-keun Ha Korea Atomic Energy Research Institute Jung-kyung Moon National Institute of Crop Science, RDA

Seed Coat Cracking (SCC) consists of irregular cracks on the surface of seed coat, giving rise to serious effects on the seed quality in soybeans. However, breeding to achieve resistance to SCC has been an arduous task due to the complicated genetic behavior and environmental interactions. Thus, this study aims 1) to determine the genetic behavior and 2) map quantitative trait loci (QTLs) with epistatic effects and QTL- by-environment interactions for irregular seed coat cracking in soybeans. The seed coat cracking was determined at natural conditions and calculated three times by counting cracked seeds with a 100 seeds per plant basis. Genetic behavior was different according to resistant parent. With KS127(TTiiii: towny pubsence, black hilum) as a parent, cracking was governed by single recessive gene showing fitted to 3:1(non- cracking : cracking) ratio but two complimentary gene showing 9:7(non-cracking : cracking) ratio with KI65(ttiiii:grey pubescence, buff hilum).

A total of 10 QTLs with additive effects were identified in the four environments. Among them, three QTLs (qSCC2-1, qSCC9, and qSCC20) located on chromosomes 2, 9, and 20 showed main-additive effects explaining 9.5% to 19.4% of phenotypic variances in two environments. Across environments, 7 additive QTLs, 4 additive x environment interactions, and 2 pairs of epistatic QTLs and epistasis x environment interactions were identified for seed coat cracking. Similar to the results in individual environments, two major QTLs (qSCC2-1 and qSCC9) showed additive main effects explaining 10.5% and 6.5% of phenotypic variances, respectively and significant additive x environment (AE) interaction in 2006 Suwon. The total QTLs explained 48.0% of the phenotypic variation, with additive effects of 33.8%, epistatic effects of 7.6%, and AE and AAE of 6.6%.

Our result suggested that soybean seed coat cracking with yellow seed coat is controlled by recessive gene but genetic behavior is varied with resistant parent and both additive effects and AE interactions can serve as important genetic basis for seed coat cracking.

Poster Number: 56 Genome-wide Mapping of NBS-LRR Genes and Their Association with Disease Resistance in Soybean

Yang Jae Kang Seoul National University Kil Hyun Kim Seoul National University Sang Rae Shim Seoul National University Min Young Yoon Seoul National University Suli Sun, Moon Young Kim, Kyujung Van, Suk-Ha Lee, Seoul National University

R genes are a key component of genetic interactions between plants and biotrophic bacteria and are known to regulate resistance against bacterial invasion. The most common R proteins contain a nucleotide-binding site and a leucine-rich repeat (NBS- LRR) domain. Some NBS-LRR genes in the soybean genome have also been reported to function in disease resistance. In this study, the number of NBS-LRR genes was found to correlate with the number of disease resistance quantitative trait loci (QTL) that flank these genes in each chromosome. NBS-LRR genes co-localized with disease resistance QTL. The study also addressed the functional redundancy of disease resistance on recently duplicated regions that harbor NBS-LRR genes and NBS-LRR gene expression in the bacterial leaf pustule (BLP)-induced soybean transcriptome. A total of 319 genes were determined to be putative NBS-LRR genes in the soybean genome. The number of NBS-LRR genes on each chromosome was highly correlated with the number of disease resistance QTL in the 2-Mb flanking regions of NBS-LRR genes. In addition, the recently duplicated regions contained duplicated NBS-LRR genes and duplicated disease resistance QTL, and possessed either an uneven or even number of NBS-LRR genes on each side. The significant difference in NBS-LRR gene expression between a resistant near-isogenic line (NIL) and a susceptible NIL after inoculation of Xanthomonas axonopodis pv. glycines supports the conjecture that NBS- LRR genes have disease resistance functions in the soybean genome.The number of NBS-LRR genes and disease resistance QTL in the 2-Mb flanking regions of each chromosome was significantly correlated, and several recently duplicated regions that contain NBS-LRR genes harbored disease resistance QTL for both sides. In addition, NBS-LRR gene expression was significantly different between the BLP-resistant NIL and the BLP-susceptible NIL in response to bacterial infection. From these observations, NBS-LRR genes are suggested to contribute to disease resistance in soybean. Moreover, we propose models for how NBS-LRR genes were duplicated, and apply Ks values for each NBS-LRR gene cluster.

Poster Number: 57 Repression of Jasmonic Acid Dependent Defenses by Aphis glycines Via Modulation Of Foliar Fatty Acid Composition

Charles Kanobe Iowa State University Jessica Hohenstein Iowa State University Gustavo Macintosh Iowa state university

The soybean aphid, Aphis glycines, is one of the main soybean pests in the Midwest. Despite the progress that has been made in elucidating the molecular mechanisms underlying plant defense against herbivory, the interaction between plants and specialized phloem feeders, such as aphids, still remains poorly understood. It has been hypothesized that aphids can block effective defense responses by blocking jasmonate (JA)-dependent signaling. To test this hypothesis, we investigated the effects of soybean aphids on the expression of wound/JA induced defenses in soybean. We show that transcription of two JA-regulated genes, PIN2 and GH3, was significantly repressed in aphid-infested and wounded soybeans when compared to wounded but uninfested plants. A similar result was obtained when JA was externally applied to aphid-infested soybeans. To gain insights on the mechanism of suppression of defenses, we performed an analysis of the fatty acid (FA) composition of plants under the same treatments, and found that in the treatments where repression of the wounding and JA responses occurred, i.e. aphids and aphid+ wounded, there was an increase in the content of 16:0 FA with a corresponding decrease in polyunsaturated FAs (18:2 and 18:3). A time course experiment revealed that differences in 18:3 content between infested and uninfested plants become evident 1 day after infestation but become statistically significant 7 days later. While the role of 16:0 in this interaction is not known, we hypothesize that the reduction in 18:3 may block the JA defense response pathway by minimizing the amount of precursors available to initiate the biosynthesis of JA. Reduced JA biosynthesis could explain the reduced response to wounding observed in aphid-infested plants. However, other mechanisms of suppression may still exist, since aphids are also able to block JA-responses when JA is exogenously applied. Therefore, we showed the aphids avoid induction of JA mediated defenses by multiple mechanisms, one of which involves hijacking the plants’ fatty acid metabolism, reducing the production of polyunsaturated fatty acids that feed the oxylipin pathway.

Key words: Jasmonic acid, Aphis glycines, fatty acids, soybean

Poster Number: 58 Transcriptome Variation between Wild Type and a Five-Foliate Leaf Mutant in Glycine max

Navneet Kaur University of Illinois, Urbana-Champaign Lila Vodkin University of Illinois, Urbana-Champaign

Molecular characterization of leaf development has not been well studied in soybean. Two soybean isolines that differed in leaf phenotype were profiled by high throughput RNA and small RNA sequencing. A Clark isoline, homozygous for a dominant mutant allele, Lf1, that specifies a five-foliate compound leaf was compared to wild type Clark that is homozygous for the standard allele that produces trifoliate leaves. Although Lf1 is dominant, it presents variable expressivity as the young plantlets with the Lf1Lf1 genotype initially have trifoliate leaves in the first few weeks, after which they transition to five-foliate leaves. At later developmental stages, they begin to produce four-foliate or trifoliate leaves. In RNA-Seq experiments, a total of 56 and 59 million reads were generated in each lane of Illumina sequencing for the vegetative bud of wild type and five-foliate-mutant libraries, respectively. Of these, 45.8 (81.7%) and 47.9 (81.1%) million reads aligned to the 78,743 target Glyma models from the reference soybean genome (cv. Williams 82) with maximum of 3 mismatches and up to 25 alignments. The comparative studies of the transcript profiles of the wild-type versus mutant line revealed a number of differentially expressed genes. A total of 15 and 94 genes were up-regulated in the wild type and five-foliate mutant, respectively, that showed >=2-fold expression difference in vegetative bud. In small RNA analysis, a collection of 200,447 and 268,508 unique small RNA sequences isolated from shoot tip tissue of wild type and mutant five-foliate were aligned to the soybean reference genome and their target glyma models were predicted using bioinformatics. This small RNA analysis at genome level reveals differences in size distribution of classes in the wild type and mutant, with the mutant five-foliate having greater number of unique small RNAs of the sizes between 21-nt to 25-nt compared to wild type. A comparison of small RNA expression of unique small RNA reads reveals higher number of the unique small RNAs (107,080) over-expressed in mutant than wild type (44,869). This study provides insight into the initial understanding of leaf development in soybean by revealing a number of genes and small RNAs differentially expressed between the wild type and five-foliate mutant. Perhaps some of these genes or small RNAs may be candidates for regulating compound leaf development in soybean.

Poster Number: 59 MSH1 Directed Epigenetic Reprogramming Modifies Maturity Time and Influences Plant Architecture in Soybean

Sunil Kumar Kenchanmane Raju University of Nebraska Lincoln Ying-zhi Xu University of Nebraska Lincoln Ajay Sandhu Sally Mackenzie University of Nebraska Lincoln

MUTS HOMOLOG 1 (MSH1) is a nuclear encoded dual targeted protein that localises in mitochondria and chloroplast nucleoids. RNAi directed suppression of this gene triggers remarkable developmental changes that are not only stable and heritable but also independent of the RNAi transgene. Comparative analysis of MSH1 suppression across plant species has revealed similar developmental reprogramming (MSH1-dr) of the plant, which includes leaf variegation, reduced growth rate, male sterility, dwarfing, altered leaf and flower morphology and changes in branching. Crossing transgene-null MSH1-dr lines with wild type isoforms produces phenotypic variation in plant growth architecture, vigor and agronomic performance. We have developed soybean F2 populations from crosses between T8 (transgene-null MSH1-dr line of Thorne) x wildtype (Thorne) and a reciprocal cross involving wildtype (Thorne) x T9 (transgene- null). Both populations were grown under green house conditions and scored for 15 agronomic traits. Significant variation was found in growth vigor, plant height, leaf length/width ratio, days to flowering, maturity time, pods per plant and seed yield. The delay in flowering time and maturity is associated with higher pods per plant and changes in trifoliate leaf shape. Data from these populations will be presented, as well as preliminary observations from field populations and F3 families being tested under green house conditions to access heritability of the variation.

Poster Number: 60 The Rpp3-Mediated Signaling Network: From Recognition to Resistance against Asian soybean Rust

Mandy Kendrick Iowa State University Michelle Graham USDA-ARS Steve Whitham Iowa State University John Hill Iowa State University Terry Graham, The Ohio State University; Reid Frederick, USDA-ARS; Kerry Pedley, USDA-ARS

Asian soybean rust (ASR) is a foliar disease caused by the obligate biotrophic fungus Phakopsora pachyrhizi, which is found in all major soybean-growing regions of the world. Commercial soybean cultivars with ASR resistance are unavailable, leaving fungicide application as the single preventative measure against ASR outbreak. Presently, six Resistance-to-Phakopsora pachyrhizi loci (Rpp1 to 6) have been mapped in soybean, and recent data implicate EDS1 and PAD4 defense pathways in Rpp2- mediated resistance. However, the genetic mechanisms and signaling pathway(s) governing ASR resistance, from pathogen perception to active defense responses, are still largely unknown for each Rpp gene. In this study we investigate ASR resistance conferred by the Rpp3 gene, which culminates in the formation of reddish-brown (RB) lesions on the soybean leaf surface following P. pachyrhizi infection. We are employing a multi-faceted approach, including gene sequence analysis, gene expression studies, map-based cloning, virus-induced gene silencing, and mass spectrometry, to deduce the Rpp3-mediated resistance pathway from pathogen perception to the RB lesion formation. Deciphering the ASR resistance pathway(s) will increase our understanding of obligate biotrophic fungi-plant interactions and is important for developing soybean lines that harbor durable resistance to Asian soybean rust.

Poster Number: 61 Developing a Transient Expression System to Study Soybean Disease Resistance Genes

Ryan Kessens Indiana University Tom Ashfield Indiana University Roger Innes Indiana University

We are studying two soybean (Glycine max) resistance (R) genes, Rpg1b and Rpg1r, which confer resistance to Pseudomonas syringae strains expressing the effector proteins AvrB and AvrRpm1, respectively. An important tool that we, and many other plant pathologists, routinely use to study R genes is Agrobacterium-mediated transient gene expression. The most commonly used plant species for transient gene expression is Nicotiana benthamiana. The problem we have encountered with N. benthamiana is that expression of AvrB or AvrRpm1 alone, without co-expression of the corresponding soybean R genes, results in a hypersensitive defense response (HR), indicating the activity of endogenous R genes. This makes it difficult to study Rpg1b and Rpg1r activity in this species. In order to identify a suitable alternative transient system to replace N. benthamiana, we have screened 15 species of Nicotiana along with 39 accessions of N. tobacum for lack of response to AvrB and AvrRpm1. This was accomplished by transiently expressing the effectors and looking for genotypes that did not display signs of HR such as tissue browning or leaf collapse. From this initial screen, we identified 6 species (N. glutinosa, N. knightiana, N. nudicaulis, N. rotundifolia, N. sylvestris, and N. tomentosiformis,) that did not respond to either effector protein (no HR) while all of the other species, and all accessions of N. tobacum, responded to one or the other. We are now using the GUS reporter gene to investigate the transformation efficiency achievable with these species to confirm they are expressing the effector transgenes at a high enough level for detection. Our ultimate goal is to reconstitute the Rpg1b/r disease resistance pathway (s) by co-expressing the soybean R genes with their corresponding effectors and any other genes that might be required for disease resistance. Encouraging results have been obtained using N. nudicaulis in which Rpg1r mediated resistance to AvrRpm1 has been observed through co-expression of both genes.

Poster Number: 62 Molecular Mapping of Soybean Rust Resistance in Soybean Accession PI 561356 and SNP Haplotype Analysis of the Rpp1 Region in Diverse Germplasm

Ki-Seung Kim Crop Sciences, University of Illinois at Urbana-Champaign Jair R. Unfried TMG - Tropical Melhoramento & Genética, Ltda. Rodovia Celso Garcia Cid, Km 87 - Caixa Postal 387, Parque Industrial, 86183-600, Cambé, Paraná, Brasil David L. Hyten Pioneer Hi-Bred, Johnston, IA 50131, USA Reid D. Frederick USDA-ARS, Foreign Disease-Weed Science Research Unit, 1301 Ditto Avenue, Fort Detrick, MD 21702, USA Glen L. Hartman USDA-ARS, Crop Sciences, University of Illinois; Randall L. Nelson. USDA-ARS, Crop Sciences, University of Illinois; Qijian Song. USDA-ARS, Beltsville Agricultural Research Center; Brian W. Diers. Crop Sciences, University of Illinois

Soybean rust (SBR), caused by Phakopsora pachyrhizi Sydow, is one of the most economically important and destructive diseases of soybean [Glycine max (L.) Merr.]. Deployment of rust resistance genes in soybean can be an effective management tool, however because of the virulence diversity in the pathogen, the discovery of novel SBR resistance genes is needed. The first objective of this study was to determine the mode of inheritance and map the location of SBR resistance gene or genes in soybean PI 561356. The second objective was to identify SNP haplotypes within the region where resistance from PI 561356 maps. One-hundred F2:3 lines derived from a cross between PI 561356 and the susceptible experimental line LD02-4485 were genotyped with genetic markers and phenotyped for resistance to P. pachyrhizi isolate ZM01-1. The segregation ratio of lesion types, reddish brown:tan, in the population supported that resistance was controlled by a single dominant gene. The gene was mapped to a 1 cM region on soybean chromosome 18 corresponding to the same interval as Rpp1. A haplotype analysis of diverse germplasm across a 213 kb interval that included Rpp1 revealed 21 distinct haplotypes of which four haplotypes were present among five SBR resistance sources that have a resistance gene in the Rpp1 region. Four major North American soybean ancestors belong to the same SNP haplotype as PI 561356 and seven belong to the same haplotype as PI 594538A, the Rpp1-b source. There were no North American soybean ancestors belonging to the SNP haplotypes found in PI 200492, the source of Rpp1, or PI 587886 and PI 587880A, additional sources with SBR resistance mapping to the Rpp1 region.

Poster Number: 63 DNA Methylation Landscape of the Soybean Genome

Kyung Do Kim Institute for Plant Breeding, Genetics and Genomics, The University of Georgia, Athens, GA 30602 Mukesh Jain National Institute of Plant Genome Research, Aruna Asaf Ali Marg, New Delhi 110067, India Scott Jackson Institute for Plant Breeding, Genetics and Genomics, The University of Georgia, Athens, GA 30602

Cytosine methylation is a common form of DNA modification in eukaryotes and plays an important role in the regulation of gene expression. Recent studies in Arabidopsis thaliana have reported that genes methylated in transcribed regions are highly expressed, while genes methylated in promoter regions show a high level of tissue- specific expression. With the development of next-generation sequencing technologies, a high-throughput approach based on bisulfite conversion has recently been available for genome-wide analysis of DNA methylation. Here, we present a DNA methylation map of the entire soybean (Glycine max) genome at single-base-pair resolution. With whole-genome bisulfite sequencing by the Illumina HiSeq 2000 platform, we detected methylated cytosines in different sequence contexts (CpG, CHG and CHH) and analyzed their distribution along each chromosome. Furthermore, the levels of DNA methylation were calculated in both transcribed and promoter regions of genes and compared with soybean transcriptome data. Our data show the methylation profiles of the entire genome and will give a better insight into the epigenetic mechanism in soybean.

Poster Number: 64 Tracing Back the History of Soybean by Estimating the Divergence Time between Glycine max And G. soja Based on Whole Genome Low Coverage Sequencing Data

Sue Kyung Kim Seoul National University Yang Jae Kang Seoul National University Min Young Yoon Seoul National University Moon Young Kim Seoul National University Kyujung Van, Suk-Ha Lee, Seoul National University

Because of the domestication process of soybean, there have been major phenotypic chances in seed size, pod length, and plant architecture. As soybean (Glycine max) is known for its high nutritional value of oil and protein, soybean has been domesticated and cultivated by one specific character trait based on human selection at a particular place over different periods of time. Importantly, tracing back in time where Glycine max and G. soja, the undomesticated ancestor of G. max have diverged plays an important role in studying of genetic diversity and in investigating the common ancestor of soybean. Ancestral divergence was analyzed via molecular clock calculations by retrieving the value of the nonsynonymous substitution mutation rate among the whole genome sequence of soybean genotypes. In this study, we sequenced 6 G. max and 6 G. soja using Illumina’s Hiseq 2000 with a low coverage sequencing technology to estimate the divergence of times between genotypes and populations. A total of the 12 genotypes were sequenced at the average depth of 6.5 and resulted 892.5 Mb and 903.3 MB consensus sequences with the coverage of 91.54% and 92.65% for G. max and G. soja, respectively. The whole genome SNP analysis showed that G. max had lower frequency levels of polymorphism (~0.1%) than G. soja (~0.25%). And, a high number of SNPs located in introns were found among 6 G. soja genotypes as SNPs were approximately twice than those found in 6 G max. The number of SNPs in G. max intronic regions was 53,134, whereas a total of 133,329 SNPs were discovered in G. soja introns. Almost an equal number of SNPs were discovered in 5’UTR and exon regions; however, different numbers of SNP in CDS and 3’ UTR were identified. By the rate of nonsynonymous change, divergence of time between G. soja and G. max would be investigated.

Poster Number: 65 Genomic Organization, Phylogenetic Classification and Differential Expression of Kunitz Trypsin Inhibitors in Soybean [Glycine max (L.) Merr.]

Won-Seok Kim Agricultural Research Service, U.S. Department of Agriculture Hari Krishnan Agricultural Research Service, U.S. Department of Agriculture

Monogastric animals fed on raw soybean meal perform poorly when compared to those fed with steam-heated soybean meal. One major contributing factor for the poor growth performance of monogastric animals is the presence of the Kunitz trypsin inhibitors (KTI) in the soybean meal. To overcome this problem Theodore Hymowitz and his colleagues at the Illinois Agricultural Experiment Station developed the Kunitz soybean variety by crossing Williams 82 and PI 157440, a KTI mutant. The Kunitz soybean variety was considered superior to commercial varieties since it lacks the abundant KTI. In this study we have compared the KTI activity between Williams 82 and Kunitz and found that the later exhibited substantial amount of trypsin inhibitor activity even though it was a KTI mutant. Western blot analysis revealed that KTI accumulated both in the embryonic axis and the cotyledons. In contrast, KTI was not detected in the embryonic axis of Kunitz. Interestingly, KTI accumulation was detected in the cotyledons of Kunitz, albeit at lower levels when compared to Williams 82. An examination of the soybean genome sequence revealed the presence of 27 KTI related genes. These genes are located on chromosomes 1, 3, 8, 9, 12, 16, 18 and 19. Of the 27 KTI genes, 14 of them were clustered on chromosome 8. Sequence analysis indicated that a vast majority of the KTI genes lack introns. In contrast, eight KTI genes contain a single intron while a single KTI gene presented two introns. Phylogenetic reconstruction based amino acid sequence homology allowed the classification of KTI into seven distinct groups. To better understand the role of the 27 KTI genes and their contribution to the seed trypsin inhibitor activity, the mRNA expression levels were measured in different organs of soybean. This analysis enabled the identification of KTI genes that were abundantly expressed in certain organs. Several of the KTI genes were expressed in developing soybean seeds. A comparative RT-PCR analysis of the KTI genes in the embryonic axis and cotyledons of the developing soybean seeds from Williams 82 and Kunitz revealed some of the KTI genes are up-regulated in Kunitz, which presumably are responsible for the residual trypsin inhibitor activity. We propose that elimination of trypsin inhibitor activity in soybean seed will require the simultaneous suppression of several KTI genes in a seed-specific manner.

Poster Number: 66 Efficient DNA Extractions of Soybean-Seed Chips for High-Throughput and Large Scale SNP Genotyping

Zachary King The University of Georgia Zenglu Li and H. Roger Boerma

In applied soybean breeding programs, marker-assisted selection (MAS) has become a necessity to pyramid value-added traits controlled by multiple genes in a single cultivar. In order to facilitate an improved MAS pipeline, the goal of this work was to create a reliable, inexpensive, high-throughput DNA extraction protocol for soybean seed chips, leaf punches, and bulk-seed samples that does not generate hazardous waste. A semi- automated seed chipper was designed and built at the University of Georgia and is capable of chipping 96 seeds at one time by slicing approximately one-tenth of the seed. By modifying, and streamlining existing protocols, the newly developed DNA extraction protocol requires approximately half the time and reagents of existing protocols and works well for all tissues listed above. Additionally, the DNA extraction platform allows for the leverage of robust SNP genotyping platforms such as the SimpleProbe Assay (Roche Applied Science) and KASPar v4.0 SNP Genotyping Systems (KBioscience) to genotype thousands of seeds nondestructively in a single day. Furthermore it is possible to run up to 100 SNP markers on the DNA extracted from a single seed chip.

Poster Number: 67 Genetic Diversity by Simple Sequence Repeat Variations among the Wild Soybean, Glycine soja, Accessions from South Korea and Other Countries

Ja-Hwan Ku National Institute of Crop Science Seok-Kee Lee National Institute of Crop Science Jung-Kyung Moon National Institute of Crop Science

Wild soybean (Glycine soja Sieb. and Zucc.) is an important source of genetic variation for introducing useful traits into cultivated soybeans [Glycine max (L.) Merr.]. Genetic diversity and differentiation among the wild soybean collected from south Korea, China, Japan, eastern Russia and Taiwan have been analyzed by simple sequence repeat (SSR) variations. Towenty-one SSR markers were used to estimate genetic diversity among 1,911 wild soybean accessions from South Korea (1,748), China (76), Japan (72), eastern Russia (11), and Taiwan (3).

The number of effective alleles varied from a low of 22 to high of 44 with an average of 29.6. When the genetic diversity was converted to population differentiation, the SSR alleles showed an average of 0.88. The wild soybean populations from South Korea, China, and Japan had high genetic diversity with indexes of 0.882, 0.861, and 0.861, respectively. In the subpopulation from South Korea, the subpopulation from Kyeonggi- Do had highest genetic diversity (0.878) and the subpopulation from Jeju-Do had lowest genetic diversity (0.832).

Poster Number: 68 Evaluation of Forage Yield and Quality for Wild Soybean Accessions and Progenies Derived from a Cross Glycine soja X G. max

Eun Ja Lee Gyeongsangbuk-do Agricultural Research & Extention Services Hong Jip Choi Gyeongsangbuk-do Agricultural Research & Extention Services Dong Hyun Shin Division of plant Biosciences, Kyungpook National University, Daegu, Korea Chan Ho Kwon Department of Animal Science & Biotechnology, Kyungpook National University, Sangju, Korea J. Grover Shannon, University of Missouri-Delta Center, P.O. Box 160 Portageville, MO 63873, USA; Jeong Dong Lee, Division of plant Biosciences, Kyungpook

Soybean is desirable as a forage crop because of it has high protein and oil concentration. Wild soybean, a progenitor of cultivated soybean, has a softer stem and higher protein content in seed than cultivated soybean. There is little information on yield and forage quality for wild soybean and its derivatives. The objective of this study was to determine the forage yield and quality of wild soybeans and selected soybeans derived from a cross G. max x G. soja.

Forage yield and quality were assessed for three grain soybean cultivars, three wild soybeans and three selected lines from G. max x G. soja. Forage quality attributes such as crude protein (CP), crude fat (CF), neutral detergent fiber (NDF), acid detergent fiber (ADF), digestible dry matter (DDM), dry matter intake (DMI) and relative feed value (RFV) were determined at the R2, R4 and R6 developmental stages . Forage yield and CF were highest at stage R6 in G. max, G. soja and selected G. max x G.soja lines. CP content was similar between R2 and R4 but increased sharply after R4 and peaked at R6 in G. max and selected lines from G. soja x G. max. On the other hand, CP content was similar between R4 and R6 stage in wild soybeans. Generally, NDF and ADF were highest at stage R4 but decreased at stage R6. DDM, DMI, and RFV increased between R4 and R6. These results suggest that R6 was the optimal harvest stage to provide forage of highest quality and yield.

A study was conducted in 2011 to evaluate forage yield and quality at stage R6 in 25 lines from PI483463 (G. soja) x Hutcheson (G. max) and four cultivated grain soybeans. Hutcheson had the highest forage yield with 24.7 t/ha in fresh weight (FW) among grain soybeans. Line W11 had the highest forage yield (25.7 t/ha, FW) among G. soja x G. max selections and four other lines had similar forage yield compared to Hutcheson. Generally the 25 lines from this G. max x G. soja cross had thinner main stems and branches than cultivated soybeans. When the 25 lines were evaluated for their feed quality as per forage grade by AFGC, nine lines rated prime grade and all 25 lines were classified as forage Grade 1. Results of this study indicate crosses between wild and cultivated soybean show promise for improving soybean as a forage crop.

Poster Number: 69 Comparative Study of Quantitative Trait Loci for Partial Resistance to Phytophthora sojae in Six Recombinant Inbred Populations Sharing One Common Parent

Sungwoo Lee The Ohio State University Rouf Mian USDA-ARS Leah McHale The Ohio State University Clay Sneller The Ohio State University Anne Dorrance, The Ohio State Universtiy

The importance of quantitative or partial resistance is increasing in plant-pathogen interaction in many crops. In soybean, Phytophthora root and stem rot caused by Phytophthora sojae is one of the destructive diseases to limit economic losses around the world. This disease has been mainly controlled by single dominant gene (Rps) resistance. However, rapid change in P. sojae population emphasized the integrated use of Rps gene-mediated quantitative and partial resistance for more durable and effective defense. Due to practical difficulties, partial resistance has been less studied and quantitative resistance loci (QRL) have been limited to use in plant breeding. In soybean-P.sojae interaction, much about partial resistance is unknown; QRL have been recently identified in several literatures, but in only a few genetic sources. Thus, it is prerequisite to identify more QRL in various resistance sources and integrated all the information for advanced studies the partial resistance. The objectives were i) to identify QRL to P. sojae in six plant introductions (PIs) originating in Korea, China, and Japan, and ii) to compare the QRL to track consistency and specificity of the QRL across the PIs. Six recombinant inbred (RI) populations sharing one common parent OX20-8 were used for this approach. It is a reasonable strategy to identify and compare QRL on the same genetic background. Comprehensive findings including novel QRL and common features of QRL will be discussed in this study. This study will provide a wider view for better understanding of molecular mechanisms of partial resistance to P. sojae in soybean.

Poster Number: 70 Copy Number Polymorphism in the SCN Resistance Locus rhg1-b from PI 88788

Tong Geon Lee University of Illinois Jianping Wang University of Florida Brian Diers University of Illinois Matthew Hudson University of Illinois

The soybean cyst nematode (SCN) resistance locus rhg1-b from PI 88788 was previously mapped to a 67-kb region on soybean chromosome 18 [formerly linkage group (LG) G]. In this study we constructed a fosmid library from PI 88788 that contains genomic clones harboring the rhg1-b locus. Five candidate fosmid clones were selected and sequenced using both the Roche 454/GS FLX+ system and Illumina MiSeq. Phrap was used to assemble the reads, and the result was visualized using Sequencher. The assembled fosmid sequences indicated that the sequences of the genes in the locus were highly conserved between the SCN susceptible Williams 82 and the SCN resistance PI 88788. The gene annotation of the genetically-defined interval predicts the presence of 11 candidate genes. At least two copies of a ~ 30 kb block harboring 5 genes were discovered by fosmid assembly in PI 88788. To estimate/confirm copy number of the block in the rhg1-b locus, whole genome shotgun sequencing of a near- isogenic line (NIL) that harbors rhg1-b from PI 88788 was conducted. Pair-end sequencing of the genomic DNA was performed using Illumina HiSeq 2000 system and alignment of NIL reads to Williams 82 reference sequence was done using the program Bowtie. This alignment shows a significant increase in sequence coverage across the five gene region, indicating greatly increased copy number. Q-PCR, fiber-FISH, additional DNA sequencing and other methods are currently being used to further define this repeat region within the rhg1-b locus.

Poster Number: 71 Map-based Cloning of the Soybean Aphid-Resistance Gene Rag2

Tong Geon Lee University of Illinois Brian Diers University of Illinois Matthew Hudson University of Illinois

The soybean aphid-resistance gene Rag2 from PI 200538 was previously mapped to a 54-kb interval on soybean chromosome 13 [formerly linkage group (LG) F]. To better understand the genomic sequence in the Rag2 interval, whole genome shotgun sequencing of a near-isogenic line (NIL) which harbors Rag2 was conducted. We used paired-end sequencing of the genomic DNA sample using the Illumina HiSeq 2000 system and obtained ~ 300 million sequence reads. De novo assembly of the genome of the Rag2-carrying NIL was done and candidate genes for Rag2 were selected using BLASTN analysis. We thus developed a fosmid library that provides genomic clones with 35 to 45 kb inserts. Two fosmid clones of 42 and 45 kb were identified by PCR- based pool screening from the interval sequence and their end sequences matched a region of the reference soybean genome sequence that was not covered by the de novo assembly. The fosmid clones were sequenced using a Roche 454/GS FLX+ system. The genetic interval for Rag2 was thus completed using overlapping fosmid clones and Illumina assembly contigs. Sequence rearrangements including several deletion/insertions were revealed between the genome of the NIL carrying Rag2 and the aphid susceptible Williams 82 reference. The gene annotation of the interval containing Rag2 predicts the presence of nine candidate genes. Of these genes, a F- BOX/Leucine-rich-repeat (LRR) candidate gene is considered to be the strongest Rag2 candidate.

Poster Number: 72 A Protein of the Unknowneome Shows a Surprising Metabolic Twist

Ling Li Iowa State University Eve Wurtele Iowa State University

Understanding of how plant composition is regulated has been elusive. The factors that regulate metabolism are key to utilization of crops for improved plant composition and production of novel constituents, yet little is known concerning the mechanisms controlling how much carbon flows to oil, starch, protein and other constituents. Recently we identified a regulatory function in starch metabolism for Arabidopsis locus At3g30720 (QQS); transgenic lines with up- or down-regulated QQS expression have a normal appearance but an altered starch content (Li et al., 2009), and a transcriptome with shifts in the accumulation of specific transcripts. QQS is among the approximately 5-20% of gene models in eukaryotic genomes that encode proteins that lack sequence homology with any known motifs (POFs, proteins with obscure features) and also are species-specific (i.e., in the case of QQS, the primary sequence is recognizable in Arabidopsis ecotypes, but not identifiable in any other sequenced species, including the closely related Brassica napus). Introduction of the Arabidopsis-specific QQS gene to soybean results in decreased seed oil and carbohydrate, and increased seed protein. Thus, this species-specific POF can affect composition in a non-native species. Iterative mutant generation and transcriptomics/metabolite profiling in Arabidopsis reveal several other POFs as candidates in regulation of starch metabolism. Protein-protein interaction analysis has revealed the QQS interactors. Taken together, the data indicate QQS as a novel regulator of plant composition, and begin to reveal the skeleton of a previously undefined network in which QQS and other POFs participate.

Poster Number: 73 Overexpression of a Soybean Ariadne-Like Ubiquitin Ligase Gene Enhances Aluminum Tolerance in Transgenic Arabidopsis

Yan Li Soybean Research Institute, National Center for Soybean Improvement, MOA Key Laboratory for Biology and Genetic Improvement of Soybean (General),National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing Xiaolian Zhang, Pei Chen, Yufeng Wang, Guangnan Xing, Tuanjie Zhao, Junyi Gai*

Aluminum is one of the most important factors limiting plant growth and yield in acid soils. Among the candidate genes located within a previous mapped Al-tolerance QTL on the soybean chromosome 11, an ariadne-like E3 ubiquitin ligase gene was cloned and named as GmARI. It has 15 extons and 14 introns, encoding a predicted protein of 586 amino acids with a RBR (RING-IBR-RING) domain. The role of ariadne-like protein in plants is largely unknown. The transcript of GmARI was expressed in soybean root, stem, leaf, flower and pod with similar amount, and was up-regulated by aluminum (Al3+, pH 4.3), salt stress, drought stress, jasmonic acid (JA), indole-3-acetic acid (IAA), salicylic acid (SA) and gibberellic acid (GA3). The trend of induction was more obvious in roots than that in leaves. Overexpression of GmARI significantly enhanced the aluminum tolerance of transgenic Arabidopsis (measured as relative root growth). These findings suggest that GmARI may participate in the aluminum tolerance and other stress responses in plants by mediating the ubiquitination and degradation of the target proteins. This is the first study showing the function of the ariadne-like E3 ubiquitin ligase gene in plant response to abiotic stresses.

Poster Number: 74 Investigation of the Possible Triplet Interactions of Phytophthora sojae Effector Proteins Avr1b and Avr1k with the Soybean Resistance Protein Rps1-k and Soybean E3 ligase GmPUB1

Chunyu Liao 1Interdepartmental Microbiology Graduate Program & Department of Agronomy, Iowa State University, Ames IA 5011 Shan Li Interdepartmental Plant Biology Graduate Program & Department of Agronomy, Iowa State University, Ames IA 5011 Madan Bhattacharyya Interdepartmental Microbiology and Plant Biology Graduate Programs & Department of Agronomy, Iowa State University, Ames IA 5011

Phytophthora sojae is an oomycete pathogen which causes stem and root rot disease in soybean. Upon infection, P. sojae delivers a series of virulent effector proteins into host cells to promote the root and stem rot disease in susceptible soybean cultivars. Avr1b and Avr1k are two such effector proteins that interact with the soybean resistance protein, Rps1-k. A soybean U-box protein gene, GmPUB1-1, was identified through its interaction with the P. sojae effector protein, Avr1b, in yeast. Pull-down assays confirmed the in vitro interaction between GmPUB1 and Avr1b. Bimolecular fluorescence complementation (BIFC) experiments implicated an in vivo interaction between GmPUB1 and Avr1b. In vitro ubiquitination assays demonstrated that GmPUB1-1 possesses E3 ligase activities and mediates self-monoubiquitination. GmPUB1 interacts with both Avr1b and Rps1-k. We hypothesize that two soybean proteins, Rps1-k and GmPUB1, together interact with effector proteins, Avr1b or Avr1k. A yeast three-hybrid system was conducted to test this hypothesis. The data of the triplet interaction study suggest that a triplet interaction most unlikely occurs among (i) Rps1-k, (ii) GmPUB1-1 and (iii) a P. sojae effector protein, either Avr1b or Avr1k. We conclude that the strong binding of GmPUB1-1 to the effector proteins makes the Ubox protein unavailable for interaction with Rps1-k.

Poster Number: 75 Enhanced Biological Nitrogen Fixation and Phosphorus Efficiency through Stabilizing Phosphate Homeostasis in Legumes

Hong Liao State Key Laboratory for Conservation and Utilization of Subtropical Agro- bioresources, Root Biology Center, South China Agricultural University Lu Qin, Jing Zhao, Jiang Tian, Xing Lu

Legume nitrogen (N) fixation is the most important N source in agro-ecosystems, but is also a process that requires a considerable amount of phosphorus (P). Phosphorus is a key non-renewable nutrient limiting world agricultural productivity. To meet the huge demands for N and P, nearly 100 million tons of N and 40 million tons of P fertilizers are used in agriculture every year. Therefore, developing legume varieties with both effective N fixation and efficient P utilization could be a sustainable approach to reduce N and P fertilization. We show here that inoculation with effective rhizobial strains dramatically enhances soybean N fixation and P efficiency in the field and hydroponics. Furthermore, we identified and characterized a nodule-preferred, high affinity phosphate (Pi) transporter gene, GmPT5, whose expression was elevated in response to low P. Yeast heterogonous expression verified that GmPT5 was indeed a high-affinity Pi uptake transporter with the Km of 25 µM. Localization of GmPT5 expression based on GUS staining in soybean transgenic hairy roots and nodules showed that GmPT5 expression occurred principally in the junction area between roots and young nodules, and in the nodule vascular bundles for juvenile and mature nodules, implying that GmPT5 might function in transporting Pi from roots into nodules. Transgenic soybean plants overexpressing GmPT5 exhibited better nodulation, while the RNAi lines severely inhibited nodule growth. Through both in situ and in vitro 33P uptake assays using the transgenic soybean roots and nodules, we demonstrated that GmPT5 mainly functioned in transporting Pi from the root vascular system to nodules. We conclude that effective nodulation could simultaneously enhance N and P efficiency in soybean, but which is critically controlled by Pi homeostasis in nodules through a high affinity Pi transporter gene, GmPT5.

Poster Number: 76 Genetic Mapping of Novel Soybean Rps Genes for Phytophthora Resistance

Feng Lin Purdue University

Phytophthora root rot (PRR), caused by P. sojae, is a widespread soybean disease which results in an annual yield loss of $1~2 billion worldwide. This disease can take place on all part of soybean plants and at all stages of soybean development, especially in soils that are poorly drained or that receive excessive irrigation.

To control the disease, breeders primarily employ race-specific resistant genes which are named Rps genes which have been identified to be located at 8 loci with 14 dominant alleles. However, soybean breeders are continuously facing big challenges due to the scarcity of excellent resistant genes and lack of efficient molecular markers for Marker Assisted Selection (MAS).

Recently pathologists have identified two germplasms (PI567139B and PI567141) introduced from Indonesia that confer excellent resistance to all major P. sojae races in Indiana soybean field and could provide potentially new resources of Rps genes.

Objectives

Dissect the inheritance pattern of the resistance genes.

Map the new genes to soybean genome.

Develop molecular markers for MAS.

Conclusion

Two independently inherited dominant resistance genes were identified by genetic analysis. Further analysis demonstrated that both of them are novel Rps genes.

Poster Number: 77 Over-expression of a Soybean Nuclear Localized Type III DnaJ Domain-containing HSP40 Reveals Its Roles in Cell Death and Disease Resistance

Jianzhong Liu Zhejiang Normal University Steve Whitham Iowa State University

Heat shock proteins such as HSP70 and HSP90 are important molecular chaperones that play critical roles in biotic and abiotic stress responses. However, the involvement of their co- chaperones in stress biology remains largely uninvestigated. In a screen to identify potential positive regulators of cell death in soybean (Glycine max), we used agroinfiltration to transiently over-express full length cDNAs of soybean genes that are highly induced during soybean rust infection in N. benthamiana leaves. Along with GmMPK6, GmMKK4 and GmMKK7, a typeIII DnaJ domain-containing HSP40 (GmHSP40.1), a co-chaperone of HSP70, caused hypersensitive response (HR)-like cell death when over-expressed in N. benthamiana leaves. The HR-like cell death was dependent on MAPKKK and WIPK, because silencing each of these genes suppressed the HR. Consistent with the presence of a nuclear localization signal (NLS) motif within the GmHSP40.1 coding sequence, GFP-GmHSP40.1 was exclusively present in nuclear bodies or speckles. Nuclear localization of GmHSP40.1 was necessary for its function, because deletion of the NLS or addition of a nuclear export signal (NES) abolished its HR-inducing ability. GmHSP40.1 co-localized with SE-RED, which has been shown to be co-localized with SR33-YFP, a protein involved in pre- mRNA splicing, implying a role for GmHSP40.1 in mRNA splicing and a link between mRNA splicing and cell death. Silencing GmHSP40.1 enhanced the susceptibility of soybean plants to Soybean mosaic virus confirming its role as a positive regulator of pathogen defense. Together, the results reveal a critical role of a nuclear-localized DnaJ domain-containing GmHSP40.1 in cell death and disease resistance in soybean.

Poster Number: 78 Diterpenoid Production by Symbiotic Bacteria

Xuan Lu Iowa State University David Hershey UC-Berkeley Reuben Peters Iowa State University

The symbiotic interaction between Bradyrhizobium japonicum (USDA110) and its soybean (Glycine max) host is a tense partnership. Although shared benefit depends on plants supplying the bacteria with photosynthate in exchange for fixed nitrogen, both organisms attempt to gain the upper hand while laboring to avoid being exploited. The B. japonicum genome contains an operon that was suggested to encode a terpene biosynthesis operon. Hypothesizing that it might play role in bacterial interaction with its host, we undertook biochemical investigations of individual genes within this operon. Previously we identified two open reading frames, blr2149 and blr2150, which successively transform geranygeranyl diphosphate into kaurene. Rhizobacterial production of kaurene, an intermediate in the biosynthesis of the diterpenene derived gibberellin (GA) phytohormones, has important physiological and evolutionary implications. A genome database search identified organisms from three separate genera of rhizobacteria that contain homologous operons. These operons contain similar open reading frames occurring in a conserved order within each genome, sharing homologous intergenic regions and having highly similar open reading frames. Here we present our results within the context of understanding the evolution of GA biosynthetic pathway, identifying how secondary metabolism is used exploit the symbiotic process and improving the production of vital legume crops.

Poster Number: 79 Ligninolytic Activity of Important Soybean Fungal Pathogens

Anatoliy Lygin Universdity of Illinois at Urbana-Champaign, Crop Scince Dep. Michelle Pawlowski Universdity of Illinois at Urbana-Champaign, Crop Science Dep. Olga Zernova Universdity of Illinois at Urbana-Champaign Curtis Hill Universdity of Illinois at Urbana-Champaign, Crop Science Dep. Glen Hartman, USDA-ARS; Jack Widholm, Universdity of Illinois at Urbana-Champaign, Crop Scinece Dep.; Vera Lozovaya, Universdity of Illinois at Urbana-Champaign, Crop Scince Dep.

Success of soybean pathogens largely depends on their capacity to rapidly produce enzymes that destroy compounds invoked in the plant defense response. The cell wall polymer lignin is an important component of soybean innate resistance to fungal invasion, since it provides a non-degradable barrier preventing entry of non-pathogenic organisms or inhibiting colonization by most soybean pathogens. We previously showed that the SDS-causing fungus Fusarium virguliforme aggressively degraded lignin in soybean roots during infection and colonization, which may be one of the primary means the fungus uses to invade soybean roots. Since this study, the ability of important soybean pathogens to degrade lignin was tested by using polymeric aromatic dye substrates to screen fungal species capacity to degrade lignin (Remazol brilliant blue R RBBR and poly R-478). The fungal isolates were grown in medium containing dyes for 2 weeks during which the fungal decolorizing ability was visually detected and a change in OD ratio was observed. Results indicated that Sclerotinia clerotiorum rapidly decolorized both dyes, but R-478 was degraded to a lesser extent than RBBR. Rhizoctonia solani was able to substantially degrade both dyes. We also tested eight different isolates of Macrophomina phaseolina (Mp) in comparison with the well-known lignin-degrading fungus Polyporus tulipifera (Pt). There were large differences in capacity to degrade aromatic dyes among the isolates with several Mp isolates showing a greater ligninolytic activity than Pt. One isolate of Mp, Conway, degraded both dyes to a greater extent compared to other isolates including isolate TN146, which when tested on plants was less aggressive than Conway. These results indicate that lignin degradation could be an important component of pathogenesis in this pathogen. Modification of lignin in soybean plants could significantly improve innate resistance to pathogens by forcing them to expend further resources towards defeating the lignin barrier.

Data on the activity of enzymes that could be potentially involved in lignin degradation will be presented.

Poster Number: 80 Engineering Resistance in Soybean to Nematodes

Benjamin Matthews USDA-ARS Soybean Genomics & Improvement Laboratory Hunter Beard USDA-ARS Margaret MacDonald USDA-ARS Sara Kabir USDA-ARS Reham Youssef, USDA-ARS; Eric Brewer, USDA-ARS

When a plant parasitic nematode attacks a host plant, the host plant induces genes to ward off the nematode while the nematode injects protein effectors into host cells to subvert the host’s defense response and to commandeer the molecular machinery of the host to increase the susceptibility of the host to the pathogen. Gene expression studies identified many soybean genes altered in expression in resistant and susceptible plant roots over time during infection by soybean cyst nematode (Heterodera glycines; SCN). However, it is difficult to assess the role and impact of these genes on resistance and susceptibility by using gene expression patterns alone. Therefore, we selected 97 soybean genes from published microarray studies and over- expressed them in soybean roots of composite plants to determine their impact on SCN development. We identified ten genes that reduced the FI by more than 50% when over-expressed. We also identified four genes that appear to enhance susceptibility more than two-fold. These studies highlight the contrasting intents of host and the pathogen during attack and provide new insights into the interactions between host and pathogen at the molecular level. Furthermore, some of these genes may be useful for genetic engineering of nematode resistance in soybean.

Poster Number: 81 SCN Phenotyping: Methods for Identifying Soybean Resistance to Soybean Cyst Nematode

Clinton Meinhardt National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211 J. Grover Shannon National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211 Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, MO 65211

Soybean cyst nematode (SCN), Heterodera glycines Ichinohe, is the most economically important pest of soybean, Glycine max (L.) Merr., in Missouri and the United States. Host plant resistance is a primary management strategy; however, there are few sources of resistance available. Most commercial varieties (greater than 90%) derive resistance from Plant Introduction (PI) 88788. Relying on PI 88788 as the main source of resistance has led to shifts in virulence and the selection of resistant SCN. Therefore, additional sources of SCN resistance are needed to better manage this pest. Soybean cyst nematode phenotyping provides an efficient process of testing soybean for resistance by comparing SCN reproduction on a test soybean plant compared to a susceptible soybean plant. Experiments are conducted according to the Standardized Cyst Evaluation 2008 protocol. Female nematodes (cysts) are counted using a fluorescence-based scanner and imaging software. This technology increases sample processing efficiency and improves data accuracy. Resistance is determined by calculating the female index. Test plants with less than 10 percent SCN reproduction compared to the susceptible plant are considered resistant. Soybean cyst nematode phenotyping is necessary to verify resistance, support the selection of breeding lines, evaluate mapping populations, and help discover new sources of soybean resistance to SCN, which will lead to the development of improved soybean varieties.

Poster Number: 82 Molecular Mapping and QTL Identification in Soybean Related To Stink Bug Resistance

Milene Moller University of Sao Paulo - ESALQ/USP Michelle Santos University of Sao Paulo - ESALQ/USP Jose Mauricio Terasawa University of Illinois at Urbana-Champaign Brian Diers - University of Illinois at Urbana-Champaign; José Pinheiro - University of Sao Paulo - ESALQ/USP

The soybean (Glycine max (L.) Merrill) is one of the most important crops in the world and is usually traded in grain, meal and refined oil, representing a major source of protein for humanity. Brazil is the second largest producer of soybean, with a production of 66 million tons of grain in an area of almost 25 million hectares, in 2011/2012. The expansion of soybean cultivation in major agricultural areas and successive planting in the same area significantly contribute to increases in the incidence of insect pests on this crop, causing losses in production. Stink bugs are considered one of the most important pests of soybean in several countries. These insects are responsible for significant losses of yield, seed quality and seed germination. They are also responsible for changes in oil and protein quantities in seeds and impair the mechanical harvesting. Aiming to control these insects, the increased use of insecticides has been frequent, causing environmental damage and raising the cost of crop production. Moreover, indiscriminate use of pesticides on certain crops has contributed to the selection of resistant insect populations. The identification of QTL in soybean related to stink bug resistance can enhance breeding programs by reducing the cycle of selection through the identification of plants with desirable traits. In this study, 174 plants from the F2 generation, obtained from crosses between IAC100 and CD-215, were used to construct a molecular map. Also, the F2:3 generation was used for phenotypic measures of 15 traits, which were combined with the molecular mapping data to identify QTL in soybean related to stink bug resistance. Linkage analysis in the JoinMap 3.0 resulted in a map with 1880 cM, consisting of 42 AFLP, 12 TRAP, 35 SSR and 341 SNP markers distributed across all 20 soybean linkage groups. The MapQTL 4.0 was used allowing the identification of QTL for all traits. The 66 QTL identified, distributed on 15 linkage groups, were significant at p<0.05. Due to a close position of some QTL we can infer that maybe the same QTL is controlling more than one trait. Those QTL associated with desirable agronomic and resistance traits will be used in a fine mapping approach to identify markers tightly linked with them. This strategy will be of greatest importance for soybean breeding programs accelerating the identification of desirable plants.

Poster Number: 83 Development of Virus-Induced Gene Silencing (VIGS) System with Soybean Yellow Mottle Mosaic Virus (SYMMV) Newly Found At Soybeans Field in Korea

Jung-Kyung Moon National Institute of Crop Science Moon Nam National Academy of Agricultura Science Jeong-Seon Kim National Academy of Agricultura Science Jae-Sun Moon Korea Research Institute of Bioscience & Biotechnology Hyoun-Sub Lim (Chungnam National University), Myung-Gun Choung (Kangwon National University), Su-Heon Lee (Kyungpook National University)

Soybean yellow mottle mosaic virus (SYMMV), a member of the genus Carmovirus, is one of the newly emerging viruses infecting soybean plants in Korea. The genome of SYMMV consists of 4,009 nucleotides of single-stranded RNA. We have constructed SYMMV virus-induced gene silencing (VIGS) clone that contain full-length cDNA copies of virus RNA which was ligated downstream of T7 and Cauliflower mosaic virus (CaMV) 35S promoter. The silencing ability of VIGS clone called as pSYMMV was tested using the soybean phytoene desaturase (PDS) gene on soybean. Partial fragment of the soybean PDS gene was inserted into pSYMMV and a transformation system was established using agrobacterium-mediated infection. Photo-bleaching of the soybean were showed in leaves in 20 days after inoculation. We identified decrease of PDS transcript levels in photo-bleached leaves of soybean using RT-PCR. Therefore VIGS system using SYMMV may be a useful tool for basic and applied studies related with gene function in soybean genome. VIGS is especially useful such as soybean that is recalcitrant to transformation. The efficient SYMMV VIGS system is an important genetic resource for breeding of soybean and has great potential as a reverse genetics tool for gene function studies in soybean.

Poster Number: 84 Phenolic compounds accumulation in soybean plants in response to Phakopsora pachyrhizi

Aguida Morales Empresa Brasileira de Pesquisa Agropecuária - Embrapa Soja Alan Alves Pereira Vicosa Federal University Lizandra Catelli Empresa Brasileira de Pesquisa Agropecuária - Embrapa Soja Aluízio Borém Vicosa Federal University Francismar Correa Marcelino-Guimarães (Embrapa Soja), Maria Cristina Neves de OliveirA (Embrapa Soja), Michelle A. Graham (USDA-ARS Corn Insects and Crop Genetics Research Unit), Clara Beatriz Hoffmann-Campo (Embrapa Soja), Ricardo Vilela Abdelnoor (Embrapa soja)

Asian soybean rust (ASR) is a soybean disease caused by Phakopsora pachyrhizi Sydow. The phenylpropanoid pathway is involved in many biological processes including the defense response to the fungus. This pathway results in the production of lignin and phytoalexins, which are an important defense against pathogens and insects. In the present work, we used high performance liquid chromatography (HPLC) to measure the production of compounds in the phenylpropanoid pathway in leaves of the resistant soybean genotype (PI459025B, Rpp4). Plants were either infected with P. pachyrhizi or mock infected with water, and leaf tissue was collected at 24, 48, 72, 96, 120, 161, 308 and 504 hours after inoculation (hai). Phenolic peak quantification was carried out by estimating the area of each detected peak from all wavelengths or their relative proportion compared to the estimated total phenolic level. ANOVA analysis followed by Tukey’s Honestly Significant Difference test was used to identify significant differences (P<.05) between treatments and time points. This study allowed identification of the conjugated isoflavones daidzin, malonyl daidzin and malonyl genistin and the isoflavones dadzein and glycitein. In addition, we identified several phenolic acids including caffeic acid, p-coumaric acid, ferulic acid and salicylic acid. The present study revealed many quantitative changes in the soluble phenolic profile of soybean in response to fungus inoculation and the accumulation of specific compounds varied over the infection time course.

Poster Number: 85 Combining Transcriptome Analyses and Virus Induced Gene Silencing to Identify Genes in the Rpp4-mediated Asian Soybean Rust Resistance Pathway.

Aguida Morales Embrapa-soja Jamie A. O’Rourke 2USDA-Agricultural Research Service, Plant Science Research Unit Aluízio Borém Vicosa Federal University Ricardo Vilela Abdelnoor Embrapa soja Kerry F. Pedley (USDA-ARS Foreign Disease-Weed Science Research Unit); Steven A. Whitham (Iowa State University); Michelle A. Graham (USDA-ARS Corn Insects and Crop Genetics Research Unit)

Six Asian Soybean Rust (ASR) resistance loci have been identified and mapped in soybean genome: Rpp1 (Resistance to Phakopsora pachyrhizi 1), Rpp2, Rpp3, Rpp4, Rpp5 and Rpp6. Of particular interest is Rpp4, which has remained stable and confers resistance against Phakopsora pachyrhizi isolates from around the world. Sequencing of the region harboring Rpp4 in the susceptible cultivar Williams 82 (Wm82) and the resistant cultivar (PI459025B) genotype identified a cluster of CC-NBS-LRR resistance genes. Meyers et al. (2009) developed Virus Induced Gene Silencing constructs from the LRR regions of the Wm82 Rpp4 candidate genes to confirm that orthologous genes were responsible for resistance in the resistant parent (PI459025B). In this study, RNA samples extracted from the same Rpp4 LRR silenced and empty vector treated plants, also infected with P. pachyrhizi (described by Meyer et al., 2009) were compared using the GeneChip® Soybean Genome Array (Affymetrix®). Since the plant samples differed only in the expression of Rpp4, comparisons of these samples would identify genes downstream of Rpp4 in the signaling pathway. In total, 383 differentially expressed probes were identified, many with functions related to defense. While the time point analyzed was late in defense signaling, bioinformatic approaches were useful in characterizing the defense response and identify transcription factors regulating the response.

Poster Number: 86 Use of Hairy Roots to Study Soybean Aphid-Soybean Interactions

Stephanie Morriss Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa Charles Kanobe Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa David Soh Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa Gregory Tylka Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa Gustavo MacIntosh, Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa

The soybean aphid, Aphis glycines (Matsumura) causes important economic losses to soybean production. There are limited natural occurrences of resistance to aphids in soybean and aphids have shown to have biotypes capable of overcoming these sources of resistance. The primary method of control is still insecticides, which are costly and have consequences to the health of humans and native insect populations. Transcriptome analyses have identified a large number of potential gene candidates for transgenic approaches that could results in plants with increased resistance to aphids. However, a high-throughput system is needed to functionally characterize these candidates and to study mechanisms of resistance as well as to identify promoters that are specifically regulated by aphids. A possible method for this high throughput approach is Agrobacterium rhizogenes transformation of cotyledons to yield hairy roots. Hairy root transformation offers a convenient alternative to transformation of soybean. Whereas transformation of whole plants requires a significant amount of time, hairy root transformation is relatively quick. However, the soybean aphid colonizes leaves and its ability to thrive on hairy roots is unknown. Thus, it is necessary to validate this system before implementation of high throughput analyses. We found that soybean aphids feed readily on hairy roots and prefer root tissue to the cotyledons used in the initial transformation. Growth analysis showed similar rates for colonies established on leaves and on roots. To further characterize the system and its potential to discriminate different levels of resistance to aphids, hairy roots obtained from resistant soybean lines carrying the Rag1 and Rag2 genes as well as a Rag1/Rag2 combination were challenged with aphids. We showed that soybean Rag1 and Rag2 resistance to soybean aphids is expressed in roots and can be detected in the hairy root system. These results support the viability of the A. rhizogenes hairy root method for studying genes involved in soybean resistance to soybean aphids. Interestingly, the only well characterized aphid resistance gene, tomato Mi-1, can also protect against root knot nematodes. We tested whether Rag1 and Rag2 could protect soybeans from infection with root knot and soybean cyst nematodes. However, and despite their expression in roots, neither gene affected nematode colonization. Our results validated a system for production of transgenic roots for examination of expression or knock-down of genes of interest in plant-aphid interactions. Poster Number: 87 Using Soybase Tools to Identify Genes Potentially Involved in Quanititative Traits: The bZIP1 Transcription Factor and Water Use Efficiency

Rex Nelson USDA-ARS CICGRU David Grant USDA-ARS CICGRU, Ames and Iowa State University, Ames, Iowa 50011 Steven Cannon USDA-ARS CICGRU, Ames and Iowa State University, Ames, Iowa 50011 Nathan Weeks USDA-ARS CICGRU, Ames, Iowa 50011 Kevin Feeley, USDA-ARS CICGRU, Ames, IA 50011; Bob Baker, USDA-ARS CICGRU, Ames, Iowa 50011; Randy Shoemaker, USDA-ARS CICGRU, Ames and Iowa State University, Ames, Iowa 50011

Understanding plant responses to stresses is crucial for crop improvement. Biological information as well as genetic and genomic data are often focused on plant models that are not in and of themselves agronomically important, but serve as starting points for experimentation in other species. Data from the model dicot Arabidopsis thaliana has been used to identify gene homologs and point to interesting questions in many other species. Recently Sun et al. (2012) identified the bZIP1 transcription factor from Arabidopsis as a positive regulator of a number of abiotic stresses that could be associated with climate change such as drought and salinity tolerance. BLAST similarity matching using the published Arabidopsis bZIP1 protein sequence against the JGI soybean gene calls indicated that there are five predicted genes with an e-value less than e-10. Using the SoyBase genome sequence browser (http://soybase.org -> Genome tab -> SoyBase browser) we identified sequenced-based genetically mapped markers that flanked the five gene models. These markers were then used to identify regions on the genetic map that harbor the identified gene calls. Examination of the genetic maps indicated that three of these genes are located within or very near QTL regions for water use efficiency (WUE) and ‘drought index’. Given that the Arabidopsis bZIP1 protein is a transcription factor, these gene models may prove to be excellent candidates as master controllers for soybean water response regulation. This exercise demonstrates the considerable power SoyBase tools provide the user to engage in cross-species comparisons and to rationally identify potential gene candidates for many quantitative traits. The gene models identified in this study and their relationships to the soybean genetic maps available at SoyBase will be presented.

Poster Number: 88 Monitoring Ds Transposition in the Soybean Genome

Hanh Nguyen Center for Plant Science Innovation, University of Nebraska-Lincoln Manmeet Singh, Shirley Sato, Saadia Bihmidine, Fareha Razvi, Truyen Quach, and Thomas Clemente

The maize two-component transposon system Ac/Ds has been used in many plant species as a means to generate insertional and activation tagged mutants. The long- term goal of this program is to develop a repository of germinal transposition events harboring mapped Ds elements that will have utility in soybean functional genomics programs. We generated approximately 500 events carrying an activation tag and 150 enhancer trap elements both delineated by Ds termini to act as initial launch sites. To investigate Ds transposition in soybean genome we stacked events harboring the activation tag or enhancer trap elements delineated by Ds termini with an Ac cassette under control of constitutive 35S CaMV promoter. We produced 587 Ac/Ds stacks carrying the activation tag and 144 Ac/Ds stacks carrying the enhancer trap. Among 17 F2 derived populations carrying the activation tagged stacks genotyped to date we observed 26 unique germinal transpositions with a germinal transposition frequency of 3.15%, whereas we found only six unique germinal transpositions from 22 F2 populations derived from enhancer trap stacks with a frequency of 0.5%. Germinal transpositions characterized to date suggest that in soybean, it appears that Ds transposes often to unlinked positions in the genome. We are currently characterizing two germinal transpositions including a enhancer trap tag in Glyma06g08110, a cyclic nucleotide binding domain gene, resulting in a phenotype with reduced pollen germination and shorter pollen tubes; and activation tag in Glyma15g21240, IMP/GMP specific nucleotidase, causing miss-expression of the tag gene. Moreover, we are currently monitoring transposition frequencies under field conditions, wherein approximately 20,000 F2 individuals from 403 F1 Ds-activation tag X Ac-transposase stack plants will be genotyped during the 2012 growing season in Nebraska.

Poster Number: 89 Identification of Wild Soybean miRNAs and Their Target Genes Response to Aluminum Stress

Hai Nian College of Agriculture, South China Agricultural University Qiao Ying Zeng College of Agriculture, South China Agricultural University Cunyi Yang College of Agriculture, South China Agricultural University Qibin Ma College of Agriculture, South China Agricultural University

MicroRNAs (miRNAs) are one type of small, endogenous RNAs that regulate gene expression by degrading target or repressing gene translation. Many miRNA play important regulatory roles in development and stress response in plants. Al toxicity is a major limitation to crop production on acid soils. Soybean is originated in China, where there are nearly 6000 wild soybeans (Glycine soja Sieb. & Zucc.) in germplasms collection. In this study, over 200 wild soybeans were collected from south China, and one Al-tolerant genotype was found and used as material to identify wild soybean miRNAs response to Al stress at the global genome-level. High-throughout sequencing and degradome sequencing were applied to sequence the libraries constructed from the root apices of this Al-tolerant wild soybean. A total of 8,616,284 and 8,712,410 primary reads in Al-treated and Al-free libraries were generated by high-throughput sequencing. We identified 104 known miRNAs and 44 new miRNAs. Among them, expression of 35 miRNAs was responsive to Al stress. Through degradome sequencing, 84 and 46 genes in Al-treated and Al-free libraries were identified as targets of miRNAs identified in this study, respectively. Gene Ontology (GO) annotations of targets transcripts revealed that 50 out of 78 targets cleaved by the eight conserved miRNA families played role in transcription regulate. Some stress related genes such as Auxin Response Factor, NB-ARC, LRR and TIR domain protein, Cation transporting ATPase, Myb transcription factors and No Apical Meristem were detected to be cleaved under Al stress. These findings provide valuable information to understand the function of miRNAs in Al toxicity and tolerance.

Key words: aluminum toxicity wild soybea high-through sequencing miRNA degradome sequencin

Poster Number: 90 Plant Resistance to Leaf-Chewing Insects: a Story of Soybean QTLs and Sharp Trichomes

Maria Ortega University of Georgia John N. All, H. Roger Boerma, and Wayne A.Parrott

Plant resistance to leaf-chewing insects reduces the need for insecticide applications, therefore diminishing production costs and pesticide concerns. In soybean, resistance to a broad range of leaf-chewing insects is found in PI229358, a soybean introduction from Japan. In PI229358, resistance is conferred by three quantitative trait loci (QTLs), which reduce plant defoliation (antixenosis) and larval-weight gain (antibiosis). From the three QTLs, QTL-M has the major effect in antibiosis and antixenosis, and is required for the expression of QTL-H (antixenosis), and QTL-G (antibiosis). The QTL-M sequence was obtained from two overlapping PI229358 BAC-clones. This region contains eleven genes, and a candidate gene was identified by comparing the sequence of resistant soybeans against the sequences of the 32 susceptible genotypes that form the US germplasm base. This candidate gene is a flavonoid glucosyltransferase that contains a SNP unique to eight insect resistant soybeans, and is absent from the susceptible soybeans. Another Japanese accession, PI227687, also exhibits antixenosis and antibiosis; however, its resistance to Lepidopteran insects is associated with the soybean gene for sharp pubescence, Pb. To determine the advantage of pyramiding the insect-resistance QTLs from PI 229358 and PI227687, near-isogenic lines of the cultivar Benning were developed through marker assisted selection. The isolines BenningQTL-M, Pb and BenningQTL-M, G, H, Pb were evaluated for their resistance to soybean looper in antixenosis and antibiosis bioassays. The experiments consisted of fifteen completely randomized blocks; each block containing a Benning, PI229358, PI227687, BenningQTL-M, BenningPb, BenningQTL-H, BenningQTL-M, Pb and BenningQTL-M, G, H, Pb plant. In the antixenosis assay, sharp trichomes significantly enhanced the effect of QTL-M in BenningQTL-M, Pb plants, which exhibited 8.5% defoliation in comparison to BenningQTL-M (24%), and Benning (46%). In the antibiosis assay, 7-day old larvae feeding on BenningQTL-M, G, H, Pb weighed an average of 7 mg, whereas larvae feeding on Benning weighed 26 mg. The results indicate that pyramiding the QTLs from PI229358 with the sharp trichome gene from PI227687 is a promising strategy to exploit their full potential towards enhancing innate resistance.

Poster Number: 91 Assembly of a Seven-Gene Stack in Soybean for the Simultaneous Production of Eicosapentaenoic Acid and the High Value Carotenoid Astaxanthin in Seed Oil

Hyunoo Park Department of Agronomy & Horticulture, University of Nebraska-Lincoln, Lincoln, Nebraska USA Fareha Razvi Center for Plant Science Innovation, University of Nebraska-Lincoln, Lincoln, Nebraska USA Paul Fraser Centre for Systems and Synthetic Biology, School of Biological Sciences, Royal Holloway, University of London, Egham, Egham, Surrey, TW200RB, UK Johnathan A Napier Deaprtment of Biological Chemistry, Rothamsted Research, Harpenden, Herts AL5 2JQ, UK Edgar B. Cahoon, Department of Biochemistry, University of Nebraska-Lincoln, Lincoln, Nebraska USA; Tom E. Clemente, Department of Agronomy & Horticulture, University of Nebraska-Lincoln, Lincoln, Nebraska USA

Consumption of fish products has rapidly increased over that past two decades. It is estimated that 50% of the world’s fish harvest will be derived from aquaculture in the near future. Of issue is the feedstocks currently relied upon for protein and oil in the aquaculture industry are wild caught fisheries, which is not sustainable. Hence, alternative, renewable feedstocks for the aquaculture industry need to be developed. To this end we have designed a prototype soybean-based feedstock for farm-raised fish. Wherein we created a seven gene stack that leads to the simultaneous accumulation of two key fish feed ingredients, namely the very long chain omega-3 fatty acid, eicosapentaenoic acid (EPA), and a high value carotenoid, astaxanthin. For the production of EPA we first created a two gene stack in soybean combining a delta 6 and delta 15 desaturase genes, respectively. A resultant event carrying this two gene stack, designated 535-9, accumulates stearidonic acid in seed oil up to 35%. We subsequently generated transgenic events that carry both delta 6 fatty acid elongase and delta 5 desaturase genes. Selected events derived from this two-gene stack have been crossed with event 535-9 to, which led EPA accumulation up to 4%. In regards to astaxanthin synthesis, we produce a set of transgenic events harboring a three-gene stack composed of the maize phytoene synthase, along with the Brevundimonas sp. ß- carotene hydroxylase (crtZ) and ß-carotene ketolase (crtW) genes. Transgenic soybean seed carrying this triple stack produced up to 800µg beta-carotene and 25 µg astaxanthin per gram seed. To create the seven-gene stack a cross of the four-gene EPA stack with the three-gene astaxanthin stack was made resulting in the production of long chain omega-3 fatty acids ranging from 3-5% in the oil, along with astaxanthin and ß-carotene accumulation ranged from 23 to 44ug/gr and 527 to1139 ug/gr in seed, respectively.

Poster Number: 92 Various Arabidopsis Root Transformation Assays Using Agrobacterium tumefaciens

So-Yon Park Univerisity of Missouri Stanton B. Gelvin Purdue University Zhanyuan Zhang University of Missouri

Root transformation assay is one of the most effective methods to study the mechanism of Agrobacterium transformation. In this study, a tumorigenic strain, Agrobacterium tumefaciens A208 (three different concentrations; 105, 106 and 107cfu/ml), was used to induce crown gall tumors in the roots of 264 Arabidopsis knock-out mutants for a root transformation screening. These mutants were selected based on 4 different categories: pathogenesis, coexpression with Agrobacterium related genes, and genes either induced or reduced after bacterium infection. Stable root transformation was conducted on the mutants using A208 before the numbers of tumors in each plant were counted. Among 264 mutants tested, 33 mutants showed elevated levels of root transformation efficiency (more than 1.5 fold increased) whereas 15 mutants displayed lower levels of transformation efficiency (50% less than wild type). Specifically, #34 and #34 interacting gene mutants (#22, #37, #155 and #156) enhanced root transformation. These genes were related to defense response to bacterium and highly expressed after bacterium inoculation. However, these mutants did not display difference from wild-type control plant in transient transformation assay using GV3101 with pBISN1 (GUS). Based on these results, we speculate that #34, #22, #37, #155 and #156 genes inhibit Agrobacterium-mediated T-DNA transformation process at host genome integration stage for plant self-defense. Further research will focus on testing these genes and their applications for improving soybean transformation.

Poster Number: 93 Fine Mapping and Identification of Candidate Genes in Linkage Group O Controlling the Resistance to Southern Root-Knot Nematode in PI96354

Anh Pham Dep. of Crop and Soil Sciences, University of Georgia, Center for Applied Genetic Technologies, Athens, Georgia 30602 Hussein Abdel-Haleem Dep. of Crop and Soil Sciences, University of Georgia, Center for Applied Genetic Technologies, Athens, Georgia 30602 H. Roger Boerma Georgia seed development commission, Athens Georgia 30605 Zenglu Li Dep. of Crop and Soil Sciences, University of Georgia, Center for Applied Genetic Technologies, Athens, Georgia 30602

Southern root-knot nematode -RKN (Meloidogyne incognita) is the most economically important disease in soybean and other crops in the southern U.S.A. and causes significant yield loss. Among available management practices, genetic resistance is the key component for its control. PI 96354 was identified to carry a high level of resistance in both galling and egg production prevention. Two QTLs were found to be responsible for the resistance in PI 96354 including a major QTL on chromosome 10 (LG O) and a minor QTL on chromosome 18 (LG G). The objective of this study was to fine-map the major QTL on chromosome 10 to develop diagnostic markers for marker-assisted selection and understand gene function and interaction. F6 RIL lines from the cross between PI 96354 and Bossier (Mi RKN susceptible) were genotyped with genetic markers to identify recombinants around the resistant allele. Analysis of these recombinant lines positioned the resistant allele between the simple sequence repeat (SSR) markers BARCSOYSSR_10_0095 and 10_0105. This places Mi-Resistant allele on LG O to a 185-kb region of the ‘Williams 82’ genome sequence that contains 30 annotated genes, but none of those belongs to R gene families. Candidate gene analysis identified four root-specific expressed genes with cell-wall modification function that have several mutations in promoter, exon and 3’UTR. Expression analysis of these genes using qPCR showed significant difference between Bossier and PI 96354 at 3,000 and 10,000 Mi eggs inoculation doses. A collection of 30 Mi-RKN resistant and 12 Mi-susceptible PIs and cultivars from Japan, China and USA was sequenced at these two candidate gene regions. A SNP allele identified from PI 96354 was present in in all of resistant PIs and cultivar genotyped, while all of the susceptible lines possess the wild-type ‘Bossier’ allele. SimpleProbe assays were developed from these SNPs for marker-assisted selection. This study narrowed the region containing the resistant gene in PI 93654 on LG O from 1.64M to 185 kb and identified promising candidate genes for further studies to investigate the role of cell-wall modification genes in conferring Mi- RKN resistance in PI 96354.

Poster Number: 94 Metabolic Engineering of Carotenoid Production in Soybean

Emily Pierce University of Georgia, Center for Applied Genetic Technologies and Institute for Plant Breeding, Genetics, and Genomics, Athens, Georgia; *University of Tennessee Donna Tucker, Peter LaFayette, Dean Kopsell and Wayne Parrott

Carotenoids are a group of natural pigments with the ability to bioaccummulate, and provide the distinctive red, yellow, and orange color to many animal products. These pigments are produced by bacteria, fungi, algae and higher plants. The pink or red ketocarotenoids, canthaxanthin and astaxanthin, are used as feed additives in the poultry and aquaculture industries as a source of egg yolk and flesh pigmentation, as farmed animals do not have access to the carotenoid sources of their wild counterparts. This supplementation is expensive and makes up a large percentage of feed costs. Because soybean is already an important component in animal feed, production of these carotenoids in soybean would be a cost-effective means of delivery. In order to characterize the ability of soybean seed to produce carotenoids, soybean cv. Jack was transformed with the crtB gene from Pantoea ananatis, which codes for phytoene synthase, the enzyme needed for carotene production in the seed. The crtB gene was engineered together in combinations with ketolases, and hydroxylases (crtW and crtZ genes from Brevundimonas SD212, and bkt and bch genes from Haematococcus pluvialis) to produce additional carotenoids from carotene; all genes were placed under the control of seed-specific promoters. Results depend on the genes used. Preliminary HPLC results show that (i) the crtB-crtW seeds produce a range of carotenoids (beta- carotene range of 14 - 713 µg/g dw; alpha-carotene, 2 - 149 µg/g dw; phytoene, 2 - 33 µg/g dw; lutein, 1.4 - 14.4 µg/g dw); (ii) The crtB-bkt seeds also produce similar carotenoids (beta-carotene range of 47 - 320 µg/g dw; alpha-carotene, 2 -54 µg/g dw; phytoene, 4 - 36 µg/g dw; lutein, 4 µg/g dw;); and (iii) the crtB-crtW-crtZ seeds have relatively lower amounts of carotenoids (beta-carotene range of 42 -336 µg/g dw; alpha- carotene, 4 -76 µg/g dw; phytoene, 3 -21 µg/g dw; lutein, 5 -13µg/g dw;), and also produce trace amounts of violaxanthin, neoxanthin and delta-tocopherol. The accumulation of phytoene, which does not normally accumulate in plant tissues, while unexpected, could be a useful product due to its powerful antioxidant properties.

Poster Number: 95 Knockout of the Fusarium virguliforme FvTox1 Gene by Homologous Recombination Revealed that the Toxin Gene is Essential for Foliar Sudden Death Syndrome Development in Soybean

Ramesh Pudake Department of Agronomy, Iowa State University, Ames, Iowa 50011 Sivakumar Swaminathan Department of Agronomy, Iowa State University, Ames, Iowa 50011 Binod Sahu Department of Agronomy, Iowa State University, Ames, Iowa 50011 Leonor Leandro Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa 50011 Madan K. Bhattacharyya, Department of Agronomy, Iowa State University, Ames, Iowa 50011

The soil borne fungus, Fusarium virguliforme, causes sudden death syndrome (SDS) in soybean. It produces the FvTox1 toxin that has been implicated in causing foliar SDS symptoms. Here we describe targeted replacement of FvTox1 from the virulent F. virguliforme Mont-1 isolate with a hygromycin B resistance gene (hph) through homologous recombination to study its role in SDS symptom development. Approximately 40 transformants were obtained per 106 conidial spores of F. virguliforme Mont-1 when the conidial spores were co-cultivated with the Agrobacterium tumefaciens EHA105 strain carrying the recombinant binary plasmid of the fvTox1 gene. The replacement of FvTox1 with the hph gene among five randomly chosen transformants was confirmed by PCR and Southern blot analyses. Only homologous recombination-mediated integration of hph into the FvTox1 locus was observed among the analyzed mutants. As expected, fvtox1 mutants failed to produce the FvTox1 protein. The absence of FvTox1 production among the knockout fvtox1 mutants was associated with a ~3-fold reduction in chlorophyll losses and foliar SDS symptom development in soybean as compared to that by the virulent F. virguliforme Mont-1 isolate. These results strongly suggest that FvTox1 is an essential toxin for foliar SDS development in soybean.

Poster Number: 96 Fibers from Soybean Stem Residue: Gene Identification And Quantitative Trait Loci (QTL) Mapping

Yarmilla Reinprecht University of Guelph, Department of Plant Agriculture Mohammad Arif University of Guelph, Department of Plant Agriculture Evan Mann University of Guelph, Department of Plant Agriculture Vaino Poysa Agriculture and Agri-Food Canada, GPCRC Harrow Gary. R. Ablett, University of Guelph, Ridgetown Campus; Istvan Rajcan, University of Guelph, Department of Plant Agriculture; Leonardo Simon, University of Waterloo, Chemical Engineering; K. Peter Pauls, University of Guelph, Department of Plant Agriculture

The use of plant fibers in automotive parts is an attractive new market for agricultural biomass but is limited by poor performance in composite materials. The principal aim of the current study was to analyze the structure and properties of soybean stem fibers with a view of assessing their potential as reinforcing additions for composite materials for use in the automotive industry. Future selections of plant cultivars optimized for this purpose could be accelerated and simplified by information about the structural and regulatory genes that control the physical properties of plant-derived fibers. The specific objectives of this research were to identify genes that contribute to soybean fiber performance in composites, map quantitative trait loci (QTL) for fiber and composite traits and develop gene-specific markers related to those traits. Databases and microarray literature were searched for the genes involved in cell wall biosynthesis and modification. PCR primers were designed and screened with genomic DNA of parents (RG10 and OX948) of a recombinant inbred line (RIL) mapping population. Gene- specific markers for key enzymes in lignin, hemicellulose, cellulose and pectin biosynthetic pathways were developed. Fifty RILs (selected based on height/ lodging index) and parents were evaluated and tested for fiber compositional traits over two years in three Ontario locations. The composites, formulated by mixing stem fibers with a polypropylene at 20% (wt/wt) by extrusion, were injection moulded and characterized for their flexural, tensile and impact properties. The addition of soybean stem residues improved mechanical properties of the composites. Both genotype and environment had significant effects on the performance of soybean stem fibers in composites. QTL for composite and fiber composition traits were distributed across the entire soybean genome and explained up to 39% of phenotypic variability for the traits. By linkage and in silico mapping, candidate genes that play roles in plant fibre formation (GRP, CAD, COMT and COBRA) were identified. Several co-localizations or associatation of composite QTL with the fiber QTL and/or fiber genes were detected on several chromosomes. This work will allow identification of key factors in fiber quality and the development of rapid, marker-based screening methods to facilitate accelerated introgression of genes for good fiber quality into new soybean cultivars.

Poster Number: 97 Omega-3 Fatty Acid Desaturase Genes in Soybean and Validation of a Major Linolenic Acid QTL

Yarmilla Reinprecht University of Guelph, Department of Plant Agriculture Kangfu Yu Agriculture and Agri-Food Canada, GPCRC Harrow Vaino Poysa Agriculture and Agri-Food Canada, GPCRC Harrow Istvan Rajcan University of Guelph, Department of Plant Agriculture Gary R. Ablett, University of Guelph, Department of Plant Agriculture; K. Peter Pauls, University of Guelph, Department of Plant Agriculture

A high level of linolenic acid (80 g kg-1) in soybean oil is associated with the development of off-flavors and poor stability. These problems can be reduced by developing low linolenic acid level cultivars. In higher plants, the level of linolenic acid in seed oil is largely determined by the activity of omega-3 fatty acid desaturases. The RG10 mutant, that has approximately 20 g kg-1 linolenic acid, has been shown to have a major linolenic acid QTL that accounted for 72-78% of linolenic acid variability, positioned on chromosome Gm14 (B2). The objectives of this study were to characterize the omega-3 fatty acid desaturase genes and validate the major linolenic acid QTL in an independent mapping population (PI 361088B x OX948). The work sequenced and localized four omega-3 FADs (Fad3A, Fad3B, Fad3C and Fad3D) in RG10 and characterized differences between these genes in RG10 and the forms found in two soybean genotypes with normal linolenic acid levels. Fad3A gene mapped to the region of a major linolenic acid QTL on chromosome Gm14 (B2), which is the same genomic region as the previously mapped fan allele that conditions low linolenic acid content in other linolenic acid mutants and is consistent with the previous placement of a microsomal omega-3 fatty acid desaturase gene. The Fad3B gene-based marker developed previously was mapped to chromosome Gm02 (D1b) in a region containing a newly detected linolenic acid QTL and corresponds well with the in silico position of Fad3B gene sequences. The positions of both QTL associated with the Fad3A gene on chromosome Gm14 (B2), as well as the minor linolenic acid QTL associated with the Fad3B desaturase gene on chromosome Gm02 (D1b), were confirmed in the PI 361088B x OX948 verifying population. Association of these QTL with the desaturase genes, their validation in an independent population and development of omega-3 fatty acid desaturase gene-specific markers should simplify and accelerate breeding soybean cultivars with low levels of linolenic acid.

Poster Number: 98 Heterotrimeric G-proteins Regulate Soybean Nodulation

Swarup Roy Choudhury Donald Danforth Plant Science Center Sona Pandey Donald Danforth Plant Science Center

Symbiotic nitrogen fixation provides a sustainable channel for the release of nitrogen into the biosphere and accomplishes the requirement for agricultural nitrogen fertilizer. Legume species form a symbiotic relationship with rhizobia and this relationship is fashioned following the exchange of a series of signals, eventually resulting in the formation of specialized root organs, nodules. Soybean is one of the most important legume plants and the recent decoding of the soybean genome has offered opportunities to explore the molecular genetic basis of nodule formation.

Heterotrimeric G protein mediated signaling is an important aspect of transmembrane signal transduction in all eukaryotic organisms. There is ample pharmacological evidence that heterotrimeric G-proteins are involved in regulation of nodulation in legumes (1). But all genetic data on the function of G-proteins in plants is restricted to Arabidopsis and rice, to date. Recently, we have identified an elaborate G-protein family in soybean, consisting of 4 G, 4 G, 10 G and 2 RGS proteins (2-4). All G protein genes are expressed in nodules and hairy roots of soybean. Specific G-protein subunits exhibit significant changes in expression at different time points after Bradyrhizobium infection. The expression of some G-protein genes was considerably changed in supernodulating and nonnodulating soybean mutants. RNAi suppression of G-protein components results in significant reduction of nodule number and change in nodule morphology. Cross-sectional view of some G-protein silenced nodules shows that the infected cells were devoid of bacteroids. Transcript levels of some of the nodulation related genes were also significantly affected in RNAi suppressed lines. Mis-expression of G-protein genes using an ENOD promoter corroborated the results obtained with RNAi-mediated suppression of G-protein genes. More importantly, the biochemical diversity observed in some of the G-protein subunits translates into differential regulation of nodulation. These results therefore provide molecular genetic evidence that the signaling pathways mediated by G-protein play important role during soybean nodulation.

References 1. Pingret, J-L., Journet. E-P., Barker. D.G. (1998) The Plant cell 10, 659-671 2. Bisht, N.C., Jez, J.M., and Pandey, S. (2011) New Phytol. 190, 35-48 3. Choudhury, S.R., Bisht, N.C., Thompson, R., Todorov, O., and Pandey, S. (2011) PLoS One 6, e23361 4. Choudhury, S.R., Westfall, C.S, Laborde, J.P, Bisht, N.C., Jez, J.M, and Pandey, S. (2012) J. Biol. Chem. 287(21):17870-81.

Poster Number: 99 The Transcriptome of Fusarium virguliforme in Infected Soybean Roots

Binod Sahu Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Jordan Baumbach Department of Agronomy, Iowa State University, Ames, Iowa 50011 Subodh Srivastava Department of Agronomy, Iowa State University, Ames, Iowa 50011 Madan Bhattacharyya Department of Agronomy, Iowa State University, Ames, Iowa 50011

Sudden death syndrome (SDS) is a major soybean disease. In North America SDS is caused by the necrotrophic fungal pathogen, Fusarium virguliforme. There are two major components to this disease: (i) foliar SDS and (ii) root necrosis. Recently, a toxin that initiates foliar SDS has been identified. Nothing however is known about how root necrosis is initiated by the pathogen. In order to unravel the pathogenicity genes in this necrotrophic fungal pathogen, we have employed a high throughput nextgen deep RNA sequencing approach. We analyzed the transcriptomes of the pathogen in infected soybean roots, as well as in the germinating conidial spores and mycelia grown in a liquid culture medium. We observed that 13,388 of the 14,845 predicted F. virguliforme genes (90%) are expressed in germinating spores and 13,294 in mycelia (90%). In infected soybean roots, 91% of the F. virguliforme genes are expressed. Among these genes, 553 genes (4.53%) and 189 genes (1.42%) showed over 10-fold and 50-fold increase in expression in infected roots compared to their average expression levels in germinating spores and mycelia. Candidate pathogenicity genes with at least 50 fold increase in transcription in infected roots compared with germinating spores and mycelia were classified into the following eight groups based on GO Ontology: (i) metabolic, (ii) cellular, (iii) primary metabolic, and (iv) oxidation-reduction processes, (v) establishment of localization, (vi) localization, (vii) transport and (viii) transmembrane transport. A majority of the genes encode enzymes with catalytic and hydrolase activities. Hydrolases most likely play a major role in establishing the necrotrophic phase.

Poster Number: 100 Candidate Gene Identification for a Transposon-Tagged Male Sterile Female Sterile Mutant in soybean

Devinder Sandhu Univerisity of Wisconsin-Stevens Point Jordan Baumbach Univeristy of Wisconsin-Stevens Point; Iowa State University Shane Dillman University of Wisconsin-Stevens Point Jaydeep Raval, Ramesh N. Pudake, Reid G. Palmer, Madan K. Bhattacharyya

In soybean the W4 gene controls the anthocyanin pigment biosynthesis in flowers. The mutant allele, w4-m, is characterized by variegated flowers. Loss of pigment production in the w4-m mutant was shown to be due to insertion of the Tgm9 transposon in the DFR2 gene. In the w4-m system, reversion of the unstable allele from variegated to normal purple color flower would indicate transposon’s excision, and its insertion at a second locus. We identified a male-sterile female-sterile mutant as a germinal revertant among the selfed progeny of w4-m. The objectives of our investigation were i) to map the sterility mutant; ii) to determine the association between sterility and presence of Tgm9 and; iii) to clone the male-sterility, female-sterility gene. We generated an F2 population by crossing Minsoy (StSt) with the sterile mutant (Stst) in heterozygous condition. Bulked segregant analysis was used to map the gene to chromosome 16. Fine mapping of the chromosome revealed that the fertility gene is flanked by BARCSOYSSR_16_0428 and BARCSOYSSR_16_0430. Comparison of the genetic linkage map with the physical map showed that these flanking markers are only 62 kb apart and five predicted genes are present in this region. Southern blot analysis confirmed that Tgm9 co-segregated with the fertility/sterility phenotype, suggesting that the sterility was caused by Tgm9. Genome walking technique was used to reveal the insertion site of Tgm9 in the st mutant. The pools of genomic DNA of homozygous fertile and sterile soybean plants were used to construct the adaptor-ligated genomic DNA libraries. The four libraries each for homozygous sterile and fertile plants were used for the PCR with the adaptor primer and a transposon-specific primer. The band specific to the libraries of sterile plants were eluted from gel and sequenced. Sequence comparison showed that the transposon insertion occurred in a helicase gene located in the St locus. The helicase gene shows homology with the DNA/RNA helicase MER3 gene, which is a DEAD-box superfamily member and is known to play a role in crossing over during meiosis. Characterization of the fertility gene will provide vital insight on structure and function of genes involved in reproductive biology of soybean and other plants. This could also help us to manipulate reproductive mechanisms for commercial applications.

Poster Number: 101 Developmental-Stage Specific and Tissue-Specific miRNA Targets in Soybean Seeds Identified by Degradome Sequencing

Md Shamimuzzaman University of Illinois, Crop Sciences, Urbana, Illinois, 61801 Lila Vodkin University of Illinois, Crop Sciences, Urbana, Illinois, 61801

MicroRNA (miRNA) mediated regulation of gene expression has immense importance at developmental stages both in animals and plants. miRNA targets have been extensively studied in model plants such as Arabidopsis and rice using computational prediction, experimental validation, and degradome sequencing. However, the identification of miRNA targets in soybean (Glycine max) is largely unexplored. Soybean is a valuable agronomic species that accumulates large reserves of proteins and oils during seed development. In order to reveal miRNA guided gene regulation in developing seeds, we performed a transcriptome-wide experimental method called degradome sequencing to detect cleaved miRNA targets without relying on predictions or overexpression. In our study, we constructed degradome libraries from three stages of seed development (25-50 mg fresh weight early-maturation green seed; 100-200 mg mid- maturation green seed; and 300-400 mg late-maturation and dessicating yellow seed). In order to identify tissue specific miRNA targets, degradome libraries for seed coats and cotyledons were prepared separately. Sequencing and analysis of degradome libraries identify 183 different targets for 80 known soybean miRNAs. When seed coats and cotyledons were analyzed irrespective of developmental stages, we found 30 cotyledon specific and 18 seed coat specific miRNAs and 32 miRNAs prominent in both tissues. Nearly 50% of the identified targets were transcription factors. One interesting observation is that we found more miRNA targets in late maturation phase when the seed is undergoing desiccation than in the earlier stages. Furthermore, we performed RNA ligase mediated 5’ rapid amplification of cDNA ends (RLM-5’RACE) to validate four different auxin response factor genes as targets for gma-miR160. Our results contribute to a more comprehensive understanding about the miRNA guided gene regulation in soybean. The miRNAs and their targets likely play significant roles in development of the immature seed and in the functional changes that occur in the cotyledons as they transition from accumulating oil and protein reserves in the immature phase to a catabolic role during seed germination.

Poster Number: 102 Factors Affecting Gas Chromatographic Analysis of Fatty Acids in Soybean

Haiying Shi National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211 Babu Valliyodan National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211 Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211

Gas chromatography (GC) has been predominantly used for the fatty acid (FA) determination in animal and plant systems including soybean. FAME (fatty acid methyl ester) formation is the key step in sample preparation. There is no very conclusive report on various factors influencing the FA determination such as initial/minimum sample size, temperature, and length of esterification reaction, the catalyst concentration, and other factors impact on the accuracy and repeatability of the analysis. It is discovered in this study that the GC response is no longer linear when the initial sample size exceeds 50 mg/mL and the optimal esterification time is four hours. This study also found that GC peak heights of the five major fatty acids is a function of injection conditions. With an injection time of 6 seconds in splitless mode, i.e. injector was vented at 6th second; methyl palmitate was measured as 9.7% of the total fatty acids. This measured concentration was decreased to 7.5% when injection time was increased up to 54 seconds. Results presented in this report are essential in establishing efficient analytical methods for oil and other traits in soybean and other plant systems.

Poster Number: 103 Identification of Candidate Defence-related Genes to Sclerotinia sclerotiorum in Soybean: Functional Analysis in Soybean, Arabidopsis and Nicotiana benthamiana

Daina Simmonds Agriculture and Agri-Food Canada Laureen Blahut-Beatty Agriculture and Agri-Food Canada Lisa Koziol Agriculture and Agri-Food Canada Lone Buchwaldt Agriculture and Agri-Food Canada Yunfang Zhang, Agriculture and Agri-Food Canada; Bernarda Calla, University of Illinois; David Neece, USDA-ARS; Steven Clough, USDA-ARS and University of Illinois

In soybean, the wheat germin gene (gf-2.8) confers a high degree of resistance to Sclerotinia sclerotiorum, a necrotrophic fungus. The transgene product, oxalate oxidase (OxO), catalyzes the breakdown of oxalic acid (OA) to produce H2O2. To identify genes with a role in defence, microarray studies have been used to examine the changes in soybean gene expression in response to infection of transgenic (resistant) and parental (susceptible) lines. In addition, the effect of OA, a major virulence determinant of S. sclerotiorum, was evaluated by leaf infiltration with OA. Thousands of genes were found to be significantly differentially expressed in each of the two studies. To identify genes related to defence, genes were classified functionally, based on the annotation of their best sequence match in public databases. Cluster analyses identified many defence- related genes that were induced across the studies, including genes annotated as GSTs, P450s, MMPs, PR proteins, WRKYs and genes of the phenylpropanoid pathway. Verification of a defence-related role of the candidate genes by over-expression or silencing is being assessed in soybean, Arabidopsis and in Nicotiana benthamiana. The goal of this study is to identify genes with the most relevant role in defence, either to aid molecular breeding or for transgenic modification to improve disease resistance, as well as to further understand the molecular interactions that take place during the infection process.

Poster Number: 104 Identification of a Soybean Fast Neutron Mutant with Increased Homogentisic Acid and Tocochromanol Accumulation

Minviluz G. Stacey Division of Plant Sciences, University of Missouri, Columbia, Missouri Rebecca E. Cahoon Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, Lincoln, Nebraska Edgar B. Cahoon Department of Biochemistry and Center for Plant Science Innovation, University of Nebraska, Lincoln, Nebraska Shirley Sato Department of Agronomy and Horticulture, and Center for Plant Science Innovation, University of Nebraska, Lincoln, Nebraska Thomas E. Clemente, University of Nebraska, Lincoln, Nebraska; Yaya Cui, University of Missouri, Columbia, Missouri; Kristin Bilyeu , University of Missouri, Columbia, Missouri; Kerry Clark, University of Missouri, Columbia, Missouri; Cuong Nguyen, University of Missouri, Columbia, Missouri; Melanie Kessler-Mathieu, University of Missouri, Columbia, Missouri; Gary Stacey, University of Missouri, Columbia, Missouri

Soybean is a major plant source of protein and oil, can fix atmospheric nitrogen and produces important secondary metabolites beneficial for human health. The published whole-genome soybean sequence (Schmutz et al., 2010) predicts more than 46,000 protein coding genes. A major future challenge will be assigning function to each of these genes, or at least identifying critical genes necessary for agronomic improvement. We have developed a large collection of deletion mutants in the soybean cultivar Williams 82 through fast neutron (FN) mutagenesis. Our goal is to develop an efficient resource for the investigation of soybean gene function through forward and reverse genetics. Unlike transposon-tagged mutants, deletion mutants (e.g., induced by fast neutron irradiation) are not widely utilized in gene function studies because mutated genes are often difficult to identify. However, the use of whole-genome, array-based oligonucleotide comparative genome hybridization (CGH) has allowed us to rapidly identify candidate genes for a number of the mutants. For example, using CGH, we were able to confirm the published deletion of FAD2-1A in M23, an X-ray mutant used for breeding for increased oleic acid content in soybean (Alt et. al., 2005; Anai et al., 2008). Thus far, we have isolated a number of mutants that affect carbon partition, development and key seed traits. We will describe these results as well as the future potential to use our fast neutron mutant population for soybean gene discovery.

Poster Number: 105 Identification of the Arabidopsis PSS1 Gene that Confers Nonhost Resistance against Two Soybean Pathogens, Phytophthora sojae and Fusarium virguliforme

Rishi Sumit Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Binod Sahu Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Min Xu Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Devinder Sandhu Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA Madan K. Bhattacharyya, Department of Agronomy, Iowa State University, Ames, Iowa 50011, USA

Nonhost resistance (NHR) provides immunity to all members of a plant species against all isolates of a microorganism that is pathogenic to other plant species. Three Arabidopsis thaliana PEN (penetration deficient) genes, PEN1, 2 and 3 have been shown to provide NHR against the barley pathogen Blumeria graminis f. sp. hordei at the prehaustorial level. Arabidopsis pen1-1 mutant lacking the PEN1 gene is penetrated by the hemibiotrophic oomycete pathogen, Phytophthora sojae, that causes root and stem rot disease in soybean. We investigated if there is any novel nonhost resistance mechanism in Arabidopsis against the soybean pathogen, P. sojae. The P. sojae susceptible (pss) 1 mutant was identified by screening a mutant population created in the Arabidopsis pen1-1 mutant that lacks penetration resistance against the non adapted barley biotrophic fungal pathogen, Blumeria graminis f. sp. hordei. Segregation data suggested that PEN1 is not epistatic to PSS1; therefore, PSS1 must encode a new form of penetration resistance. Microscopic evaluation of pss1 leaves inoculated with P. sojae zoospores showed that pss1 but not pen1-1 or wild type Col-0 supports completion of the P. sojae life cycle suggesting complete breakdown of nonhost resistance against this pathogen in the pss1 mutant. The pss1 mutant is also infected by the necrotrophic fungal pathogen, Fusarium virguliforme, which causes sudden death syndrome in soybean. Thus, a common NHR mechanism is operative in Arabidopsis against both hemibiotrophic oomycetes and necrotrophic fungal pathogens that are pathogenic to soybean. However, PSS1 does not play any role in immunity against the bacterial pathogen, Pseudomonas syringae pv. glycinea, that causes bacterial blight in soybean. We mapped PSS1 to a region very close to the southern telomere of chromosome 3 that carries no known disease resistance genes. Identification and further characterization of the PSS1 gene would provide further insights into a new form of nonhost resistance in Arabidopsis, which could be utilized in improving resistance of soybean to two serious pathogens.

Poster Number: 107 Analysis of QTLs for Resistance to Phomopsis Seed Decay Associated with Maturity Time in Soybean (Glycine max)

Suli Sun Seoul National University Moon Young Kim Seoul National University Kyujung Van Seoul National University Yin-Won Lee Seoul National University Baodu Li, , Qingdao Agricultural University, China; Suk-Ha Lee, Seoul National University

Phomopsis seed decay (PSD), primarily caused by Phomopsis longicolla, is a major contributor to poor soybean seed quality and significant yield loss, particularly in early maturing soybean genotypes. However, it is not yet known whether PSD resistance is associated with early maturity. This study was conducted to identify quantitative trait loci (QTLs) for resistance to PSD and maturity time using a recombinant inbred line (RIL) population derived from a cross between the PSD-resistant Taekwangkong and the PSD-susceptible SS2-2. Based on a genetic linkage map incorporating 117 simple sequence repeat markers, QTL analysis revealed two and three QTLs conferring PSD resistance and maturity time, respectively, in the RIL population. Two QTLs (PSD-6-1 and PSD-10-2) for PSD resistance were identified in the intervals of Satt100-Satt460 and Sat_038-Satt243 on chromosomes (Chrs) 6 and 10, respectively. These QTLs do not overlap with any previously reported loci for PSD resistance in other soybean genotypes. Two QTLs explained phenotypic variances in PSD resistance of 46.3% and 14.1%, respectively. Among three QTLs for maturity time, two (Mat-6-2 and Mat-10-3) were located at positions similar to the PSD resistance QTLs. The identification of the QTLs linked to both PSD resistance and maturity time indicates a biological correlation between these two traits. The newly identified QTLs for resistance to PSD associated with maturity time in Taekwangkong will help improve soybean resistance to P. longicolla.

Poster Number: 108 QTL Mapping of Resistance to Fusarium virguliforme that Causes Sudden Death Syndrome (SDS) in Soybean

Sivakumar Swaminathan Department of Agronomy, Iowa State University, Ames, Iowa 50011 Nilwala Abeysekara Department of Plant Pathology and Microbiology, Iowa State University, Ames, Iowa 50011. Madan Bhattacharyya Department of Agronomy, Iowa State University, Ames, Iowa 50011 Silvia Cianzio Department of Agronomy, Iowa State University, Ames, Iowa 50011

SDS is caused by the soil-borne fungus, Fusarium virguliforme. The pathogen infects, colonizes, and causes root necrosis and rot in soybean. The pathogen has never been isolated from the diseased foliar tissues. One or more toxins produced by the pathogen have been considered to cause foliar symptoms. The disease causes significant yield reduction, which depending on genotype may be up to 100%. The estimated average annual yield loss from this disease has been valued over 100 million dollars. SDS resistant soybean cultivars provide partial resistance to the pathogen, and it has been a major method of controlling this disease. To determine the map locations of the genes encoding SDS resistance, F7 recombinant inbred lines (RILs) were screened for SDS resistance and susceptibility using three protocols: 1) infecting roots with F. virguliforme inocula prepared in sorghum meals, and 2) stem cut and 3) root feeding assays feeding soybean seedlings with crude toxin preparation from culture filtrates. Twenty highly SDS resistant lines from population AX19286 (A95-684043 X LS94-3207) were identified by applying all three protocols. Genomic DNA from individual plants was analyzed by the Illumina Golden Gate Assay for 1,530 SNPs. For the AX19286 population, one major QTL responsible for resistance to root necrosis by the pathogen was mapped to chromosome 19. A separate QTL governing tolerance of soybean to F. virguliforme toxins present in culture filtrates was found in the chromosome 13. Three additional QTL were discovered from a separate segregating population developed using the SDS resistant line, LS98-0582. Of these, one located on chromosome 20 confers root resistance and two QTL located on chromosomes 9 and 20 encode tolerance to the toxin preparations.

Poster Number: 109 QTL Identification Related to Resistance to Japanese Beetle In Soybean

Jose Terasawa University of Illinois at Urbana-Champaign Milene Moller University of Sao Paulo Brian Diers University of Illinois at Urbana-Champaign

The soybean (Glycine max (L.) Merrill) is one of the most important crops for humanity as primary source of oil and protein. The United States of America (US) cultivated 29.80 million hectares in 2011 and it is the world major producer of soybeans with a production of 83.13 million tons (USDA, 2012). The expansion of soybean cultivation in major agricultural areas and successive planting in the same area significantly increased the incidence of insect pests, challenging yield potential and causing losses in production. New pests that can economically impact the production of soybean must be focus of breeding efforts in order to reduce the use of pesticides, minimize the negative economic impact to farmers and to the economy of those countries involved in the soybean production, processing and trade. The Japanese beetle (Popillia japonica) is a Coleopteran defoliator pest that has been introduced in the US in the early twentieth century and it can survive in many plant species, including soybean. Two F3:4 populations, segregating for insect defoliation occasioned by Japanese beetle, were developed by crosses made between germplasm line HC95-15MB (resistant parent) and elite cultivars LD00-3309 (susceptible parent) and LD02-4485 (susceptible parent). HC95-15MB line has sources of insect resistance from PI229358. Choice tests were performed in 184 F3:4 lines from Population 47 (LD00-3309 X HC95-15MB) and 175 F2:3 lines from Population 48 (LD02-4485 X HC95-15MB). The experiment was conducted in controlled cages at field conditions in summer 2011 at University of Illinois at Urbana-Champaign, allowing free movement of the Japanese beetles in order to test antixenosis effect in both populations. The individuals were rated according to the percentage of defoliation. Moreover, a selective genotyping was employed using plants with lower and higher ratings for each population, consisting of 57 plants from Population 47 and 55 plants from Population 48. The parents and the selected individuals were genotyped in a 1536 SNP analysis using the Illumina Golden Gate Assay resulting in 465 and 479 polymorphic markers for each population, respectively. Statistical analyzes were performed in SAS9.3 and R/qtl. Five QTL were significant at p<0.05 for each population considering a LOD>=2 in a simple interval analysis (MLE method). Two of those QTL were identified for both populations at the same LG, emphasizing their presence considering the different background of the populations. A fine mapping approach will be used to identify markers closely linked to promising QTL.

Poster Number: 110 Low pH, Aluminum and Phosphorus Coordinately Regulate Malate Exudation through GmALMT1 to Improve Soybean Adaptation to Acid Soils

Jiang Tian State Key Laboratory for Conservation and Utilization of Subtropical Agro- bioresources, Root Biology Center, South China Agricultural University Cuiyue Liang State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, Root Biology Center, South China Agricultural University Leon V. Kochian Robert W. Holley Center for Agriculture and Health, USDA-ARS, Cornell University Hong Liao State Key Laboratory for Conservation and Utilization of Subtropical Agro- bioresources, Root Biology Center, South China Agricultural University Miguel A. Pineros, Zhufang Yao, Lili Sun, Jiping Liu, Jon Shaff

Low pH, aluminum (Al) toxicity and low phosphorus (P) availability often coexist and are heterogeneously distributed in acid soils where crops need to cope with these multiple limiting factors simultaneously for better growth. To date, the underlying mechanisms of crop adaptation to these multiple factors as a complex on acid soils remain poorly understood. In this study, we found that P addition to acid soils could alleviate Al toxicity with regards to root growth which enhanced soybean adaptation to acid soils, especially for the P-efficient genotype, HN89. Further studies in hydroponics showed that both internal malate content and malate exudation of roots were suppressed by low pH. Interestingly, compared to the P-inefficient genotype HN112, HN89 released more malate from its roots under hydroponic conditions that mimic the primary aspects of acid soils - low pH, high Al, and low P supply, suggesting that root malate exudation might be critical for soybean adaptation to acid soils more than to detoxify Al. A soybean malate transporter gene, GmALMT1, was cloned from the Al-treated root tips of HN89. Transient expression of GmALMT1 in both onion epidermal cells and soybean protoplasts showed that GmALMT1 was localized on the plasma membrane. Functional characterization in Xenopus laevis oocytes demonstrated that GmALMT1 encodes a membrane transporter that mediates malate efflux in an extracellular pH dependent manner. Like root malate exudation, GmALMT1 expression was also pH-dependent, being suppressed by low pH, but enhanced by Al plus P addition to the solution bathing the roots of HN89, which correlated with the enhanced malate exudation from roots under the same conditions. Furthermore, overexpression and knockdown of GmALMT1 in transgenic soybean hairy roots, as well as overexpressing GmALMT1 in transgenic Arabidopsis indicated that GmALMT1 mediates roots malate efflux and could help to detoxify Al. Taken together, our results suggest that malate exudation might be the critical mechanism of soybean adaptation to acid soils, which is coordinately regulated by three factors, low pH, Al and P supply, through regulation of GmALMT1. Considering the distribution of pH, available P and active Al concentration along the profile of acid soils, we speculate that GmALMT1 could regulate adaptive root architecture and malate secretion thorough carbon allocation to acid soils and thus GmALMT1 could be considered as a potential candidate gene for improving soybean yields on acid soils via biotechnological approaches.

Poster Number: 111 A Soybean Pathogenesis-Related Protein PR10.1 Plays an Important Role in the Root Nodule Symbiosis

Katalin Toth University of Missouri Jun Wang, Zhe Yan, Laurent Brechenmacher, Michael Henzl, Jeremy Dahmen, Li-juan Qiu and Gary Stacey

Root hair cells represent an attractive model for studying single cell systems biology. Different biological processes occur within the single root hair cell responding to a broad range of biotic and abiotic stimuli. Root hairs play an important role in water and nutrient uptake, as well during the interaction with beneficial and pathogenic soil microorganisms. Root hair cells serve as the entry point for infection of legume plant roots by nitrogen fixing soil bacteria (rhizobia) during the establishment of root nodule symbiosis. In previous work, our laboratory documented a variety of transcriptomic, proteomic and metabolomic changes that occur within the soybean root hair cell in response to rhizobial inoculation. Among the genes found to respond to Bradyrhizobium japonicum inoculation were those annotated as playing a role in plant defense against pathogen infection. A protein belonging to the broad protein family of plant pathogenesis-related proteins (PR10/Bet v1), GmPR10.1 was found to be up-regulated in root hair cells a few hours post inoculation. The PR10 protein family is one among 17 protein families designated as pathogenesis-related proteins that show responses not only in plant-microbe interactions, but also to biotic and abiotic inputs. Despite the wide response of these genes to a variety of stresses, the biological function of the PR10 family remains to be uncovered. Soybean transgenic roots carrying a GmPR10.1 silencing construct showed reduced nodule number without morphological alterations. Interestingly, overexpression of the GmPR10.1 protein resulted in a significant increase in nodule numbers on transgenic soybean roots inoculated with B. japonicum. It has been reported that some members of the PR10 protein family display ribonuclease activity. RNase activity could be shown for the GmPR10.1 protein in vitro. Whether this ribonuclease activity is important during the nodulation process remains to be investigated. Localization studies of GmPR10.1 labeled with fluorescent protein showed a cytoplasmic localization of the protein in Nicotiana benthamiana leaf epidermal cells. We are also investigating the extent to which root and nodule physiology is impacted by ectopic expression of GmPR10.1.

Poster Number: 112 Metabolomic Plasticity in Soybean Tissues under Water Deficits

Babu Valliyodan National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA Haiying Shi National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA Dong Xu Computer Science Department, University of Missouri, Columbia, Missouri 65211, USA Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA

Integration of soybean metabolome changes with transcriptome changes is a powerful approach to understand key processes such as growth characteristics, stress responses, and yield. The major focus of our research is to build comprehensive map of metabolites for soybean and help make connections between regulatory or metabolic pathways not previously characterized. Drought is one of the major abiotic stresses causing yield reduction in soybean. The root and shoot system plays specific roles in stress tolerance mechanisms in plants. Soybean Williams 82 was grown in controlled conditions and the water deficits were imposed by withholding water starting from three weeks after germination. Stress levels were measured and root, leaf, and stem tissues were collected at specific stress conditions; very mild stress, mild stress, severe stress, and recovery after stress along with the respective well-watered controls. We have utilized GC/MS and LC/MS platforms to identify metabolites for further characterization. Also, we compared the transcriptome changes associated with the key metabolic pathways under water deficit conditions. We have identified several tissue specific and stress specific metabolites, which help understand the biochemical networks involved in stress responses. For example, severe stress induces massive mobilization of carbon to the root system, consistent with a shift to root growth under these conditions. Also, there is a strong increase of nucleotide compounds, especially purines, which indicates the build-up of nitrogen compounds for root growth. There were early indications of carbon/nitrogen (C/N) balance alterations and synthesis of osmoprotective compounds even under very mild stress (VMS) conditions in leaf and stem, changes that magnified under mild stress (MS). These changes were not considerably greater under severe stress (SS) in leaf and shoot, suggesting that MS conditions are sufficient to initiate the stress response program in leaf and stem tissues.

Poster Number: 113 Expression Profiling Analysis of Soybean Transcription Factor Genes in Response to Xanthomonas axonopodis pv. glycines

Kyujung Van Seoul National University Kil Hyun Kim Seoul National University Yang Jae Kang Seoul National University Sang Rae Shim Seoul National University Taeyoung Lee, Moon Young Kim, Suk-Ha Lee, Seoul National University

Bacterial leaf pustule pustule (BLP) caused by Xanthomonas axonopodis pv. glycines (Xag) is a serious disease in soybean. To investigate the role of transcription factors (TFs) in plant defense mechanisms under Xag treatment, soybean near-isogenic lines (NILs) carrying BLP-susceptible and BLP-resistant allele were analyzied by RNA-seq. A total of the differentially expressed 2,415 genes were identified between the BLP NILs at 0, 6, and 12 hr after Xag infection. Using SoyDB and SoybeanTFDB, which are soybean TF databases, a total of differentially expressed 351 TF genes were identified. Top ten major TF families (AP2-EREBP, MYB, bHLH, WRKY, NAC, GRAS, C2C2(Zn) CO-like, C2H2 (Zn), TPR, and ZIM) were comprised of almost 80% from 351 TF genes. Especially, the propotions of AP2-EREBP, WRKY and ZIM TF families were more than doubled in other TF families compared with overall SoyDB compositions. We further surveyed distributions of 351 TF genes specifically up- or down-regulated in the BLP- resistant NIL compared with the BLP-susceptible NIL at each time course (0, 6, and 12 hr). Among 351 TF genes, 263 and 40 TF genes were up-regulated and down- regulated, repectively. The rest 48 TF genes were expressed by up- and down- regulation at each time period in the BLP-resistant NIL. Most of TF genes were highly up-regulated in the BLP-resistant NIL at 0 hr. Additionally, cis-regulatory elements (CREs) involving in regulationg stress-responsive transcription, ABRE, G-box, MYBR, MYCR, and W-box were investigated. A total of 1,092 downstream genes containing these CREs were identified. Our analysis suggested that substantial progress of understanding how plant immunity occurs via the interactions of TFs and CRES

Poster Number: 114 Exploration of the Genes in a Quantitative Disease Resistance Locus in Soybean for Defense against Fungal Pathogens

Jinrong Wan National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA J. Grover Shannon National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA Anne E. Dorrance Department of Plant Pathology, Ohio State University/OARDC, Wooster, Ohio 44691, USA Henry T. Nguyen National Center for Soybean Biotechnology, Division of Plant Sciences, University of Missouri, Columbia, Missouri 65211, USA

Fungal pathogens (including oomycetes) cause approximately 50% of all soybean disease losses in the United States, with an average monetary loss of approximately 2 billion dollars each year. Durable and broad-spectrum resistance is needed to reduce or prevent such yield losses caused by diverse fungal pathogens. Recently, a major Quantitative Trail Locus (QTL) responsible for quantitative resistance to Phytophthroa sojae, a fungus-like oomycete organism, was mapped by our group to Chromosome 13 (Linkage group F). Considering that broad-spectrum resistance is generally conferred by quantitative resistance, this region may contain genes conferring resistance not only to P. sojae and but also to other fungal pathogens. To reveal which genes are involved in the observed resistance to P. sojae and whether these genes are also involved in defense against other pathogens, especially fungal pathogens, we have selected twenty candidate genes from this QTL region based on their annotation, expression pattern, and responsiveness to the inoculation of P. sojae and other pathogens. The genes showing differences in either sequence or expression between two parents, which were used to make the recombinant inbred lines for mapping the QTL, will be over-expressed and silenced in soybean hairy roots to infer their functions in defense against fungal and oomycete pathogens.

Poster Number: 115 QTL Analysis of Unsaturated Fatty Acids in a Recombinant Inbred Population of Soybean

Xianzhi Wang South Dakota State University Guo-Liang Jiang * South Dakota State University Marci Green South Dakota State University Roy Scott USDA-ARS David Hyten, USDA-ARS; Perry Cregan, USDA-ARS

Soybean [Glycine max (L.) Merr.] is an important oilseed crop which produces around 30% of the world’s edible vegetable oil. The quality of soybean oil is determined by its fatty acid composition. Vegetable oils with high-oleic and low-linolenic acids are desirable for human consumption and other uses. The objectives of this study were to identify quantitative trait loci (QTLs) for unsaturated fatty acids, and to evaluate the genetic effects of single QTLs and QTL combinations in soybean. A population of 87 recombinant inbred lines (RILs) derived from the cross of SD02-4-59 x A02-381100 was evaluated for unsaturated fatty acid content in seven environments. A total of 516 polymorphic single nucleotide polymorphism (SNP) markers and 298 polymorphic simple sequence repeat (SSR) markers were used to genotype the mapping population. By using composite interval mapping (CIM), inclusive composite interval mapping (ICIM) and/or the interval mapping (IM) method, a total of seven QTLs for oleic, six QTLs for linoleic and six QTLs for linolenic were detected in two or more environments. Two major QTLs for oleic acid on linkage group E (Chromosome 15) (qOLE-E) and G (Chromosome 18) (qOLE-G-2), two major QTLs for linoleic on linkage group A1 (Chromosome 5) (qLLE-A1-2) and G (qOLE-G-1), and three major QTLs for linolenic on linkage group D1b (Chromosome 2) (qLLN-D1b), F (Chromosome 13) (qLLN-F) and G (qLLN-G) were consistently detected in at least three individual environments and the average data over all environments. Significant QTL x QTL interactions were not detected. However, significant QTL x environment interactions were detected for most QTLs. Some QTLs reported previously were confirmed, and some new QTLs were identified in this study. Comparisons of two-locus and three-locus combinations indicated that cumulative effects of QTLs were significant for all the three unsaturated fatty acids. QTL pyramiding by molecular marker-assisted breeding would be an appropriate strategy for improvement of unsaturated fatty acids in soybean.

Poster Number: 116 Overexpression of -Expansin Gene GmEXPB2 Improves Phosphorus Efficiency in Soybean

Xiurong Wang South China Agricultural University, China Jia Zhou South China Agricultural University, China Jianna Xie South China Agricultural University, China Hong Liao South China Agricultural University, China

Soybean (Glycine max) is an important oil crop in agricultural production, but low phosphorus (P) availability limits soybean growth and development. Expansin is a family of plant cell wall proteins, which involves in a variety of physiological processes, including cell division and enlargement, root growth and leaf development. In this article, a soybean -expansin gene GmEXPB2, which was significantly induced by phosphate (Pi) starvation, was successfully overexpressed into soybean plants. Constitutive overexpression of GmEXPB2 promoted leaf development with 33.5%, 62.1% and 45.2% increases in leaf area, and root growth with 20.3%, 20.0% and 34.0% increases in root surface area in three independent transgenic plants, and consequently resulted in improved P efficiency in OE-6 and OE-23 transgenic plants, which exhibited 106.9% and 115.1%, 130.5% and 134.3%, and 103.4% and 87.5% increases in plant dry weight, P content, and seed weight compared to wild-type plants under P-limited conditions in hydroponics, respectively. In particular, two GmEXPB2 transgenic soybean lines showed increased plant dry weight by 30.5% and 26.8%, and P content by 53.2% and 40.2% to wild-type plants in low-P calcareous soils. These findings indicate great potential for improving soybean production on low-P soils. To our knowledge, this is the first report in which improvement of P efficiency was achieved through constitutive overexpression of an endogenous EXPB gene in soybean.

Poster Number: 117 Pinpointing the Gene Underlying A Major QTL For Southern Root-Knot Nematode Resistance By Whole Genome Resequencing

Xiangyang Xu Division of Plant Sciences and National Center for Soybean Biotechnology (NCSB), University of Missouri, Columbia, Missouri 65211 Liang Zeng Beijing Genome Institute, Main Building, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China Ye Tao Beijing Genome Institute, Main Building, Beishan Industrial Zone, Yantian District, Shenzhen, 518083, China Tri Vuong Division of Plant Sciences and National Center for Soybean Biotechnology (NCSB), University of Missouri, Columbia, MO 65211 Jinrong Wan, Roger Boerma, Jim Noe, Zenglu Li, Steve Finnerty, M.D. Pathan, Grove Shannon and Henry T. Nguyen

Southern root-knot nematode, Meloidogne incognita, is an important parasitic nematode of soybean in southern United States. The objectives of this study were to develop a sequencing-based genotyping approach, dissect QTL for root-knot nematode resistance into individual genes, reveal the landscapes of SNP distribution and recombination patterns, and gain insight into soybean genome evolution. Two hundred forty six recombinant inbred lines (RIL) derived from a cross between Magellan and PI 438489B were evaluated for southern root-knot nematode resistance in a greenhouse and sequenced at an average of 0.19 X coverage. A method based on maximum parsimony of recombination (MPR), Bayesian inference and a hidden Markov model was utilized to analyze the sequence data generated from the RIL population. Based on 109,273 phased SNPs, a total of 3509 bins were defined and a bin map was subsequently constructed. About 9.1% of bins contain a single gene, and 41.7% of them harbor 2 to10 genes. Three QTL for southern root-knot nematode resistance were identified, and the major QTL was mapped to the bin 10 of chromosome 10, which is 29.7 Kb in size and harbors three true genes and two pseudogenes. Sequence variations were detected in two candidate genes encoding cell wall-modifying enzymes, and the gene underlying the major QTL for southern root-knot nematode resistance was identified. Our results suggested that the sequencing-based genotyping approach greatly enhance QTL mapping accuracy. Also, a comprehensive characterization of genome-wide recombination patterns was conducted, and genomic features associated with meiosis recombination were identified.

Poster Number: 118 Responsiveness of Soybean CBF and COR genes to cold stress

Yuji Yamasaki Indiana Univ Purdue Univ Indianapolis Stephen Randall Indiana Univ Purdue Univ Indianapolis

Environmental stresses such as cold, drought, and salinity limit plant growth and in the worst case do not allow plant survival. Stress responsiveness in cold tolerant plants is contributed by two different major pathways, the ABA dependent pathway and the ABA independent pathway which utilizes AREB and CBF 1, 2, & 3 in Arabidopsis. During cold stress, the transcription factors in these pathways are strongly up-regulated and induce expressions of COR genes (for example, some dehydrins). Soybean is a cold intolerant and not able to acclimate to cold stress. In this report, we demonstrate the ability of soybean to respond to cold by up-regulating transcript of SCOF-1 and CBF. Expected downstream targets of these transcription factors, such as dehydrins, are not transcriptionally responsive, nor do dehydrin proteins accumulate. Other predicted target transcripts such as ADH, LEA14 and galactinol synthase likewise do not accumulate in response to cold treatments. We are currently testing functionality of the soybean CBF genes in Arabidopsis by evaluating COR gene expression in Arabidopsis.

Poster Number: 119 Genome-Wide Identification of Soybean Root Hair microRNAs that Respond to Bradyrhizobium japonicum Infection

Zhe Yan Divisions of Plant Science and Biochemistry, National Center for Soybean Biotechnology, Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO Oswaldo Valdés-López, Jixian Zhai, Marc Libault, Jun Wang, Trupti Joshi, Laurent Brechenmacher, Seth Findley, Lijuan Qiu, D. Janine Sherrier, Blake C. Meyers, Dong Xu, and Gary Stacey

Micro-RNAs (miRNAs) are single-stranded RNA molecules 21 to 24 nucleotides in length. Plant miRNAs negatively regulate the expression of endogenous genes and play important roles during plant developmental processes. To investigate the role of miRNAs during soybean nodulation, three small RNA libraries were generated and sequenced using the Illumina platform. The small RNA libraries were derived from root hairs and stripped roots (i.e., roots without root hairs), mock inoculated or inoculated with Bradyrhizobium japonicum. Sequencing of these libraries identified a total of 178 miRNAs, including 47 novel miRNAs. Comparing miRNA abundances among libraries, 32 miRNAs were detected only in stripped roots, while 12 miRNAs were detected only in root hairs. The expression levels of 93 miRNAs were differentially regulated (>= 2- fold) by B. japonicum inoculation. A Parallel Analysis of RNA Ends (PARE) library was constructed and sequenced to reveal a total of 459 miRNA targets. By way of example, the roles of miR4397-5p, miR1514, miR4416 and TAG_32412, four B. japonicum responsive miRNAs, were further analyzed. Ectopic expression of these miRNAs in soybean roots resulted in significant changes of nodule numbers for miR4416 and TAG_32412 overexpressing lines, while no significant change was observed in miR4397-5p and miR1514 expressing lines.

Poster Number: 120 Functional characterization of a Soybean BAG Gene and Its Potential Role in Nematode Resistance

Gregory Yeckel Division of Plant Sciences, University of Missouri Pramod Kandoth, Melissa G. Mitchum

Plants resistant to the soybean cyst nematode (SCN) mount a hypersensitive cell death- like response upon nematode feeding, but the genes regulating this process are not known. Laser-assisted microdissection of nematode feeding cells coupled with microarray analysis identified a soybean gene upregulated 87-fold in plants resistant to SCN that shared sequence similarity with the Arabidopsis BAG6 (Bcl-2 associated anthogene 6) gene. BAG genes encode an evolutionarily conserved family of proteins in animals, yeast and plants. These proteins contain a conserved BAG domain which mediates interaction with the molecular chaperone HSP70/HSC70. Members of the BAG protein family in animals and yeast function in apoptosis to regulate a range of activities from inhibition to promotion of cell death. However, much less is known about the role of BAG proteins in plants. A family of seven BAG genes (AtBAG1-7) has been identified in Arabidopsis thaliana. AtBAG6 was shown to induce programmed cell death in both yeast and Arabidopsis. Here we report the functional characterization of a soybean BAG6-like gene and its potential use in engineering a novel form of nematode resistance in crop plants.

Poster Number: 121 Studying Genetic Diversity and Phylogenetic Relationship between Glycine max and G. soja for Soybean Divergence

Min Young Yoon Seoul National University Sue Kyung Kim Seoul National University Yang Jae Kang Seoul National University Ahra Bae Seoul National University Moon Young Kim, Kyujung Van, Suk-Ha Lee, Seoul National University

Soybean (Glycine max (L.) Merr.) is one of the most important crops around the world. Understanding genetic variations of wild soybean progenitor (G. soja Sieb. Zucc.) is crucial because of the useful genetic property as a natural gene pool to overcome the insufficient genetic backgrounds of the cultivars. Domestication process of cultivated soybean from wild soybean in Asia is obscure meaning, but more clear evidences are needed for improving soybean in breeding program. After 104 wild and landrace soybeans from China, Korea and Japan were selected, a total of 89 SSR primers, which cover 20 chromosomes as averaging 28 alleles per SSR locus, were used for genotyping of 104 lines. After population was confirmed by AMOVA and the STRUCTURE software version 2.3, the identified SNPs of chloroplast DNA (cp DNA) sequences between wild (IT182932) and cultivated (PI 437654) soybean were surveyed with the population. Variations based on cp DNA SNPs were used for construction of phylogenetic tree using MEGA 4.0 program. In this research, genomic DNA genotyping and cp DNA SNPs detection could be differentiated from the existing approaches for genetic variations between G. soja and G. max as well as it offers valuable information to examine domestication and evolutionary process. Based on the genetic diversity analysis of SSR dendrogram using 104 wild and landrace soybeans, a total of 12 soybean genotypes (6 G. max and 6 G. soja) were selected. Using next-generation sequencing technology of Illumina GAII, genome sequencing of these 12 soybean genotypes was performed at lower sequencing coverage. The 892.5 Mb and 903.3 Mb sequences were produced from these 6 G. max and 6 G. soja with the coverage of 91.54% and 92.65%, respectively. We will compare sequence variations and haplotype diversity among soybean populations. Also, single nucleotide polymorphisms as nonsynonymous change in protein coding regions from each genotype will be identified and contributed to help understanding of the complexity of G. max and G. soja divergence.

Poster Number: 122 Natural Genetic Variation in GmAOS1 Enhances Soybean Resistance to Insect Attack

Deyue Yu Nanjing Agricultural University Hui Wang Nanjing Agricultural University Juanjuan Wu Nanjing Agricultual University Zhongjie Gao Nanjing Agricultural University

Soybean displays considerable natural variation for resistance to insects. Developing soybean insect resistance varieties would be a sustainable and economical approach to soybean production. Here a soybean allene oxide synthase encoding gene (GmAOS1) was cloned. Expression study revealed that the corresponding mRNAs were widely distributed among the stems, leaves and roots. The transcript levels of GmAOS1 increased after the common cutworm feeding and reached the highest levels after 3h treatment. GmAOS1 gene coding region show very high sequence conservation and there was only one SNP among 184 soybean accessions. However, 40 SNPs/Indels were found in the promoter regions of this gene. Association analysis suggest that GmAOS1 was associated with soybean resistance to common cutworm, and its genetic variation modulates insect resistance through reducing the growth rate of common cutworm and increasing the compensation ability of soybean. There were a 3.5-fold difference for the growth rate of common cutworm and a 1.5-fold difference for two compensation index between two of the most differentiated haplotype classes. Selection of favorable GmAOS1 alleles would enable breeders to more effectively select insect resistance mateirals in soybean.

Poster Number: 123 Fine Mapping a Soybean Aphid Resistance Gene, rag4 in Soybean (Glycine max L.)

Jiazheng Yuan Michigan State University Cuihua Gu Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824 Umesh Rosyara Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824 Qijian Song USDA-ARS, Beltsville, MD 20705, USA Perry Cregan, USDA-ARS, Beltsville, MD 20705, USA; Decun Wang, Department of Crop and Soil Sciences, Michigan State University, East Lansing, MI 48824

The soybean aphid (Aphis glycines Matsumura) has become a major pest on soybean [Glycine max (L.) Merr.] after it was first found in the United States in 2000. The application of chemicals for the pest management is expensive and leads to the adverse environmental impact. Possible innate genetic sources of soybean resistance to the aphid exist in the plant introductions (PIs) and US cultivars. The soybean aphid resistance in PI 567541B was controlled by two genes (Mensah et al., 2008), rag1c and rag4, that were later mapped to the soybean chromosome 7 (LG M) and chromosome 13 (LG F), respectively and these two genes explained 31-88% of the phenotypic variations in the trials (Zhang et al. (2009). The QTL underlain the rag4 region was previously positioned between the SSR markers Satt348 and Satt649 (7.46 mbp in- silico distance) on soybean chromosome 13 (Zhang et al., 2009). To rapidly confirm and narrow down the genomic interval of the rag4, various approaches were employed to finemap the rag4 region in this study. The Illumina® 6k HD beadchip and our customer designed Taqman® and Kaspar® SNP markers were used to genotype our mapping and finemapping populations [two recombinant inbred lines (RILs), several high generation residual heterozygous lines (RHLs) and a BC3F3 population]. In turn, the QTL underlain rag4 was positioned between MSUSNP13-5 and ss247923149. Furthermore, several recombination breakpoints were identified in several lines and these recombination events displayed a significant segregation of the soybean aphid resistant trait. Based on these recombination events in these lines, we have fine- mapped the rag4 gene between SNP marker MSUSNP13-29 and MSUSNP13-31 within 162135 bp interval.

Poster Number: 124 Divergent Patterns of Endogenous Small RNA Populations from Seed and Vegetative Tissues of Glycine max

Gracia Zabala University of Illinois Lila Vodkin University of Illinois Edhilvia Campos, Kranthi K. Varala, Sean Bloomfield, Sarah I. Jones, Hlaing Win, Jigyasa H. Tuteja, Bernarda Calla, Steven J. Clough, Matthew Hudson

A collection of 135,055 unique small RNA sequences isolated from eight tissue samples representing two stages of immature seeds and vegetative parts of young soybean plants were mapped to the soybean genome and its predicted cDNA gene models. Bioinformatics and other analytic tools were used to distinguish miRNAs, siRNAs and tasiRNAs, as well as their genomic origins and potential target genes. Tissue-differential expression based on the flux of normalized miRNA and siRNA abundances in the eight smRNA libraries was evident, some of which was confirmed by smRNA blotting. The global view of these smRNA populations also revealed that the size classes of smRNAs varied amongst different tissues, with the developing seed and seed coat having greater numbers of unique smRNAs of the 24-nt class compared to the vegetative tissues of germinating seedlings. The 24-nt class is known to be derived from repetitive sequences including transposable elements. Detailed analysis of the size classes associated with ribosomal RNA genes and transposable element families, showed greater diversity of smRNAs in the 22- and 24-nt size classes but with the 22-nt siRNAs having higher number of sequence reads. We will present specific examples of differentially expressed gma-miRNAs and the genes they target for silencing. Also, putative silenced genes and the siRNA collections complementing them, in some instances, in a tissue specific manner. We will present evidence for the existence in G. max of ortholog genes to the Arabidopsis thaliana TAS3 genes as well as the miRNAs that target the gma-TAS3 genes. Supported by United Soybean Board and Illinois Soybean Association.

Poster Number: 125 Small RNAs Related to Ribosomal RNA Genes found in Seed and Vegetative Tissues of Glycine max Complemented Genes with Multiple Annotation

Gracia Zabala University of Illinois at Urbana-Champaign Lila Vodkin University of Illinois at Urbana-Champaign Navneet Kaur

Among the 135,055 unique small RNA sequences isolated from eight tissue samples representing immature seed and vegetative parts of young soybean plants, a significant number mapped to more than 25 different locations in the soybean genome. One class of those small RNAs were siRNAs that complemented regions of the soybean genome on chromosomes 13, 15 and 16 that contain multiple repeats of 26S, 18S and 5.8 S ribosomal RNAs. The data showed that these regions were a source of siRNAs with a broad size range which can be generated by any of multiple mechanisms such as the one involving the enzyme complex (RDR2/DCL3) utilized to generate the 24-nt siRNAs that silence repeated transposable elements in Arabidopsis. The sizes of the most abundant unique small RNA sequences generated by these (26, 18, 5.8)S rRNA repeats were 21 to 24-nts. One of the repeated regions first identified in Gm15 has homology to GenBank clone, FJ980442.1, annotated as 18S rRNA gene, partial sequence; internal transcribed spacer 1, 5.8S rRNA gene. A different repeat found first in Gm16 is a 26S ribosomal RNA insertion in a serine-threonine protein kinase family member gene. Those two sequences found in Gm15 and Gm16 are propagated multiple times in Gm13 within at least 10 large Glyma gene models. We determined that the current Phytozome soybean genome does not remove or annotate these ribosomal DNA regions correctly but shows them as having Glyma gene models with varied annotations that predict peptide domains. The overall percentage of this ribosomal RNA-siRNA class ranged from a few percent in each library to a maximum of about 10% in the germinating cotyledon library. Interestingly, RNA-seq data obtained from shoot tips of three G. max isolines (Clark standard-wild type; Clark glabrous-hairless mutant; Clark foliate-five leaflet mutant) showed higher expression of the 26S ribosomal RNA containing Glyma-gene models in chromosome Gm13 in the foliate-mutant shoot tips compared to wild type and glabrous lines. This differential expression could be the result of a less effective silencing of the repeated ribosomal RNA sequences that should correlate with a decrease in complementary ribosomal RNA-siRNAs. Supported by United Soybean Board and Illinois Soybean Association.

Poster Number: 126 Genetic Engineering of Soybean Plant Innate Resistance

Olga Zernova University of Illinois at Urbana-Champaign Anatoliy Lygin University of Illinois at Urbana-Champaign Curtis Hill, UUC, Crop Science; Glen Hartman, USDA-ARS; Jack Widholm UUC, Crop Science; Vera Lozovaya, UUC, Crop Science

Plant resistance is an economical and sustainable disease management option. In higher plants, complete resistance genes have helped effectively control certain diseases caused by the pathogens which are specialists, exhibiting host specialization. For some other diseases, caused by generalist pathogens, complete resistance does not exist and partial resistance has mostly been ineffective under field conditions, probably because the pathogens causing these diseases have the capacity to avoid recognition by the host plant. In addition, the innate resistance in most soybean genotypes may not be strong enough to limit colonization of these pathogens. Efforts to increase the strength of the innate defense system would help limit colonization by these pathogens.

One essential part of the innate immune system is the production of secondary metabolites which are toxic to many pathogens when they either preexist (phytoanticipins) or are rapidly produced (phytoalexins) in effective concentrations and in appropriate plant tissues after pathogen attack.

Non-native phytoalexins may enhance plant broad-spectrum resistance, because pathogens may not have developed the capacity to detoxify non-host phytoalexins.

To improve management of pathogens in which no complete resistance exists genetic engineering provides tools via increase or expression of the biosynthesis of the natural anti-microbial compounds in different plant tissues.

The capacity of pathogens to metabolize and detoxify phytoalexins may also be a factor that is linked to the pathogenicity and/or virulence of some pathogens. Understanding of mechanisms of enzymatic detoxification of phytoalexins by pathogens may open new prospects for developing tools of plant disease control. With this knowledge, researchers will design antifungal agents selective against specific pathogens or clone fungal genes controlling phytoalexin detoxifying enzymes to use to silence these genes in plants. Such novel tools may work synergistically with the natural phytoalexins to provide more effective and environmentally safer methods for controlling phytopathogens and protect crops.

Data will be presented demonstrating effects of soybean non-native phytoalexin resveratrol on important soybean pathogens and response of soybean transgenic plants accumulating resveratrol to fungal infection.

Poster Number: 127 Isolation and Characterization of ‘GmScream’ Promoters which Regulate the Highest Expressing Soybean (Glycine max) Genes

Ning Zhang Department of Horticulture and Crop Science, OARDC/The Ohio State University Leah McHale Department of Horticulture and Crop Science, OARDC/The Ohio State University John Finer Department of Horticulture and Crop Science, OARDC/The Ohio State University

Soybean contributes the largest transgenic percentage acreage of any crop. The transgene primarily consists of the gene of interest, along with a promoter, enhancer and terminator, which together regulate the gene of interest. The promoter may be the most important component of gene regulation and largely determines the level and specificity of gene expression. With the generation of novel soybean transgenics for both basic and applied research, it is important to identify new promoters from soybean, which could be used to regulate various transgenes of interest. Moreover, the identification of novel promoters is especially important for stacked genes, which require different promoters to regulate each coding region. However, only a few soybean promoters have been isolated and characterized, and little is known about the regulatory elements of soybean promoters. In this study, 1.5 kb upstream regions were identified and cloned from a series of highly expressed genes. The promoters were placed upstream of the green fluorescent protein (GFP) gene in standard vectors (pFLEV, Finer Laboratory Expression Vector) used for direct DNA introduction via particle bombardment and in binary vectors (pCAMBIA1300) used for Agrobacterium- mediated transformation. Constructs harboring the promoter:GFP fusion were evaluated using transient expression with lima bean cotyledonary tissues and stable expression in soybean hairy roots. Most of the GmScream promoters showed higher GFP expression than a constitutive CaMV35S (Cauliflower Mosaic Virus 35S) promoter in the two expression systems. The strongest GmScream promoter that we have cloned yielded about 5.5-fold higher expression than CaMV35S promoter while several showed 2- to 3- fold higher expression. In this research, we have identified some novel soybean promoters and evaluated promoter strength using two rapid validation tools. These strong soybean promoters should be useful for both basic research and soybean improvement. Moreover, regulatory elements within those strong promoters will be identified and selected for further analysis. We expect to generate a range of strong native soybean promoters as well as identify the elements that contribute to high levels of gene expression.

Poster Number: 128

Interactions between a Nematode, Fungus and Aphid: Implications for Soybean Management

Michael McCarville Entomology, Iowa State University, Ames, Iowa Matthew O'Neal Entomology, Iowa State University, Ames, Iowa Gregory Tylka Plant Pathology, Iowa State University, Ames, Iowa Gustavo MacIntosh Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa

Soybean is an introduced crop to America, because of this it has benefited from a small number of pests threatening its production. Since its rapid expansion in production acres beginning in the 1930s, several pests have been introduced from the native range of soybean. Our knowledge of how these pests interact and the implications for management is limited. We examined how three common economic soybean pests, the soybean cyst nematode (SCN), the brown stem rot fungus (BSR) and the soybean aphid (SBA) interact on soybean. From 2008 to 2010 six soybean cultivars were exposed to four different pest treatments consisting of SCN, BSR and SBA in a micro- plot field experiment. Pest treatments were manipulated by using artificial infestations in a field virgin to soybean production. Plots were artificially infested with either a single pest or all three pests in combination. The performance of each pest was measured in a “single pest” treatment and compared to pest performance measured in the “multiple pest” treatment. This designed allowed us to measure the impact of the presence of two other soybean pests on the performance of each pest. Soil samples were taken prior to planting and after harvest to assess SCN performance during the season. Internal stem discoloration was used as a measure of BSR performance. Soybean aphid populations were monitored from initial infestation to leaf senescence to track performance. The presence of multiple pests significantly decreased SBA and BSR performance, but significantly increased SCN performance.

Poster Number: 129

Transgenerational Variation of Gene Expression during the Innate Immunity Response of Soybean

Oswaldo Valdes-Lopez Divisions of Biochemistry and Plant Sciences, National Center for Soybean Biotechnology, C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA Robert J Schmitz Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 92037, USA.; Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 92037, USA Saad M Khan Computer Sciences Department, C. S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA Shiqi Cui Department of Statistics, University of Missouri, Columbia, MO 65211, USA Jing Qiu Department of Statistics, University of Missouri, Columbia, MO 65211, USA Trupti Joshi Computer Sciences Department, C. S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211, USA Brian Diers Department of Crop Sciences, University of Illinois, Urbana, Illinois, 61801 Glen L Hartman United States Department of Agriculture-Agricultural Research Service, University of Illinois, Urbana, Illinois, 61801 Joseph Ecker Plant Biology Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 92037, USA.; Genomic Analysis Laboratory, The Salk Institute for Biological Studies, La Jolla, CA, 92037, USA.; Howard Hughes Medical Institute, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, CA, 92037 Gary Stacey Divisions of Biochemistry and Plant Sciences, National Center for Soybean Biotechnology, C.S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211

Microbe associated molecular pattern (MAMP)- triggered immunity (MTI) is an important component of the plant innate immunity response to invading pathogens and its magnitude can vary across different plant species and even among cultivars. We previously demonstrated that the variation in pathogen resistance observed in two soybean parental lines (LD0-2817 and LDX01-165) was inherited to the progeny. We hypothesize that the variation in gene expression observed in the same parental lines might be inherited. To prove this, we analyzed the transcriptional profile of both parents and two RIL lines (one with strong MTI response and one with a weaker response) by RNAseq technology. The results indicate that 70% of the up-regulated genes (fold- change > 2) were commonly regulated among the two parents and the two RIL lines; contrary to this, only 1% of the down-regulated genes (fold-change < -2) were commonly regulated among these four genotypes. Likewise, we observed that the parent LD0-2817 and the RIL line 11272 (both showing a strong innate immunity response) exhibited more pronounced gene expression changes (at least 2 fold- change) compared to the parent (LDX01-165) and the RIL (11268), which have a weaker innate immunity response. These results suggest that the variation in gene expression observed in the parental lines is inherited to the progeny. Additionally, with this analysis we identified around 70 genes that can be used to identify additional expression-QTL related to soybean innate immunity. Poster Number: 130

About the Sheraton West Des Moines…

Complimentary Local Shuttles

The hotel offers complimentary shuttles within a three mile radius. Their shuttle service runs from 7am until 11pm. The hotel has 2 vans that hold 10 people each.

Local Entertainment

Local Movie Theater Century 20 Jordan Creek & XD – Jordan Creek Mall

Local Shopping  Jordan Creek Mall and Village Shops – Including Dillard’s, Younkers, and Scheels  West Glen Towne Center – Upscale home and specialty shops, restaurants and clubs  Valley West Mall – Including Younkers, Von Maur and JC Penney

Entertainment Venues- within 3 miles  Living History Farms: a 550 Acre Open-air Museum where visitors can travel at their own pace through five historical time periods  Clive Aquatic Center with leisure pool, lap pool, waterslide, lazy river and shaded seating  Waveland Golf Course: over 100 year-old, beautifully maintained, 18-hole golf course  Jordan House: beautifully restored 1850’s Victorian home was a stop on the Underground Railroad  Funny Bone Comedy Club at West Glen Towne Center  Blue Moon Dueling Piano Bar at West Glen Towne Center Local Restaurants near the Sheraton West Des Moines

Within walking distance:

The Tavern – Pizza 1755 50th St. Palmer’s Deli - 50th St. Mi Mexico – 11407 Forest Ave 515.222.6933 Applebee’s - 11411 Forest Ave Chilis - 11410 Forest Ave Bakers Square - 1310 NW 114th – 515.224.0808

Nearby: up to 2.25 miles away

Trostle’s Dish - University 515.221-DISH Twin Peaks – 4570 University Ave 515.528.8294 Nick’s Grill and Bar – 9769 University 515.221.1338 Sam and Gabe’s Italian Bistro - 7700 University 515.271.9200 Cool Basil Contemporary Thai & Vegetarian - 8801 University, Suite 22 - 515.223.8111 Taki Japanese Steakhouse & Sushi - 2677 86th St., Suite 2601 Noodle Zoo – 6750 Westown Parkway 515.440.0411 Rock Bottom Brewery - 4508 University 515.267.890 Waterfront Seafood Market & Sushi – 2900 University 515.223.5106 Macaroni Grill - 4502 University – 515.267.8400 Ohana’s Japanese Steakhouse – 2900 University 515.225.3325 Biaggis’s Ristorante Italiano – 5990 University 515.221.9900 Outback Steakhouse – 10901 University 515.221.3309 Jason’s Deli - 4200 University HuHot Mongolian Grill - 4100 University 515.457.9090 Qdoba Mexican Grill - 10201 University Olive Garden - 3600 Westown Parkway Granite City Food & Brewery- 12801 University

At Jordan Creek Mall there are several eateries around the parameter of the mall: About 2.5 miles away

Samuri Sushi & Hibachi- 7125 Mills Civic Parkway 515.233.4888 Cheesecake Factory – 101 Jordan Creek Parkway 515.457.9888 PF Chang’s – 110 Jordan Creek Parkway 515.457.7772 Bravo! Cucina Italiano – 120 Jordan Creek Parkway 515.225.0660 On the Border Mexican - 140 Jordan Creek Parkway 515.224.6440 Joe’s Crab Shack – 130 Jordan Creek Parkway 515.226.9966 Fleming’s Prime Steakhouse-150 Jordan Creek Parkway 515.457.2916 Buffalo Wild Wings- 6925 Mills Civic Parkway 515.221.9464 Panera Bakery Café- Jordan Creek Mall Taxi

Capitol Cab Company 515.282.8111

City Cab 515.279.5555

Freedom Taxi Cab 515.289.9800

United Cab Ltd 515.277.7784

Yellow Cab Company 515.243.1111

Bus

DART (Des Moines Area Regional Transit Authority) www.dmmta.com

Des Moines Convention and Visitor’s Bureau

www.seedesmoines.com Phone 1.800.451.2625

Sheraton West Des Moines