COMPARATIVE GENOMICS OF LEGUMES

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0151715
• Project No.: MIN-22-015
• Project Start Date: Oct 1, 2008
• Project End Date: Sep 30, 2013

Project Director
Young, N.

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

Performing Department
Plant Pathology

Non Technical Summary
Crop legumes play a critical role in agriculture as important sources of nutritional protein and oil, forage for livestock animals, raw materials for biofuels, and beneficial compounds in human health. Legumes are also unique in their ability to fix atmospheric nitrogen, a property that is important both in plant nutrition and environmental quality. Nitrogen fixation results from a novel symbiotic relationship between legume roots and soil bacteria known as rhizobia. My lab is interested in the genes that control how legumes interact with other living organisms, both beneficial ones like symbiotic rhizobia as well as disease causing pathogens. Previously, we developed genetic mapping resources for locating and characterizing disease resistance genes, primarily in soybean. These resources have proven especially useful as tools for marker-assisted breeding. More recently, research in the lab has shifted to genome sequencing as a framework for gene discovery and the development of tools for genetic analysis and breeding. As part of this shift, we have focused on a model legume, Medicago truncatula, a close relative of alfalfa. Medicago exhibits typical legume biology but also has several characteristics that make it favorable for genetic analysis. This research has led to the nearly complete genome sequence for Medicago and the characterization of numerous symbiotic and disease resistance gene families. In the next few years, we will exploit genome sequence data in soybean and Medicago to discover new genes of agriculture importance and develop improved tools for legume improvement. Specifically, we will focus on sequence-based markers known as Single Nucleotide Polymorphisms (SNPs) that provide exceedingly powerful capacity to identify and isolate genes of interest. We will begin by discovering many thousands of SNP markers in M. truncatula and then translate this technology to alfalfa. In M. truncatula, we will use SNPs primarily to map and clone genes that control the capacity of legumes to form symbiotic relationships with rhizobial bacterial. In alfalfa, we will use SNP markers to develop enhanced disease and stress tolerance along with increased biomass. In soybean, large numbers of SNP have already been described. We will use these SNPs to create a large relational database that integrates the genetic marker data for Minnesota adapted germplasm together with results of field and greenhouse experiments. In the process, we will develop the capacity to identify pairs of soybean lines that are most favorable as parents for new breeding populations, as well as faster and more efficient markers for selecting disease resistant cultivars during the course of the breeding program.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
212	2410	1040	10%
212	2410	1080	10%
212	2499	1040	10%
212	2499	1080	10%
212	1820	1040	30%
212	1820	1080	30%

Knowledge Area
212 - Pathogens and Nematodes Affecting Plants;

Subject Of Investigation
2499 - Plant research, general; 1820 - Soybean; 2410 - Cross-commodity research--multiple crops;

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

Keywords

legumes

medicago

alfalfa

soybean

genome sequencing

single nucleotide polymorphism

snp

association mapping

marker assisted breeding

disease resistance

symbiosis

Goals / Objectives
GOAL 1. Translate the Medicago truncatula genome sequence to alfalfa. We will discovery SNPs in M. truncatula and alfalfa through genome re-sequencing as a basis for characterizing genes involved in plant-microbe interactions, both beneficial and harmful. Next generation sequencing (454 and Solexa) enable cost effective re-sequencing, given a reference genome sequence. We have collaborated with Joann Mudge and Greg May at the National Center for Genome Research in a series of pilot re-sequencing experiments using a second M. truncatula accession, discovering approx. 160,000 tentative SNPs with 75 percent validated. Once discovered, M. truncatula SNPs can be used to assay haplotype structure of ecotypes. To carry out the high density SNP assays, we will use Illuminas Bead Station recently acquired by the Minnesota Agricultural Experiment Station. It enables a wide range of sample throughput and broad experimental scale. The M. truncatula genome sequence can also act as a reference for SNP discovery in alfalfa. M. sativa has an autotetraploid genome structure that makes genome analysis difficult, so alfalfa SNPs and association mapping requires M. truncatula as a starting point. This will present novel bioinformatic challenges, as we will need to recognize changes resulting from speciation versus those that result from variation within alfalfa. The outcomes will be: 1. Discovery of new SNP markers useful for association mapping and haplotype analysis in M. truncatula and alfalfa and 2. A relational database consisting of SNP markers for both M. truncatula and M. sativa and useful in characterizing genome organization, genome diversity, and association mapping. GOAL 2. Create a soybean SNP database for use with Minnesota breeding germplasm In soybean, SNPs enable better marker-assisted selection (MAS), association mapping, and strategic choice of parents to use in crosses. We will expand our use of SNPs in the breeding program and generate a relational database to integrate molecular marker and haplotype data with field and greenhouse data. Preliminary research indicates that LD extends long genomic distances in soybean as a result of recent shared ancestry. Consequently, genome-wide SNP scans of Minnesota germplasm provides the breeding program with a genome snapshot for each line, including shared and contrasting haplotypes. This makes possible two avenues of research. First, we can implement deterministic selection of parents carrying desirable haplotype combinations. Second, we can initiate discovery of new gene trait associations by comparison of phenotype with SNP haplotyping. In both cases, we will start by targeting disease resistance. For this, we will choose 384 lines from the soybean breeding program and carry out genome-wide scans using the Bead Station system described above. The outcomes will be: 1. Better markers for screening disease resistant soybean lines, 2. Combinations of molecular markers for use in choosing pairing of parents that yield the most favorable combinations of haplotypes, and 3. Novel associations between genome regions and quantitative disease resistance.

Project Methods
Overview. Re-sequencing and SNP discovery will focus on M. truncatula and M. sativa, as there are already more than 2000 characterized SNPs in soybean. In Medicago, we will initially characterize SNP frequency, distribution, and decay of LD, an essential parameter for association mapping. Later, we will expand to association mapping, targeting QTL associated with symbiosis. In soybean, SNP analysis will target Minnesota germplasm associated with the disease resistance, leading to association mapping of disease resistance QTL. Re-Sequencing and SNP Discovery. To discover Medicago SNPs, we will resequence 5 accessions using the Solexa 1G platform. Each accession will be re-sequenced to 5X coverage, which translates to two Solexa runs per accession. Alfalfa is a polymorphic autotetraploid, so each sequencing run will reveal as many as four haplotypes after alignment to M. truncatula. Determining phase (which SNPs are on the same vs. separate strands) will often be impossible, but this does not limit SNP discovery. For data acquisition, we will use Illumina base-calling and filtering. Sequence data will be analyzed using Alpheus software, which provides a pipeline for aligning the millions of short reads to the M. truncatula genome as a basis for data-warehousing, filtering, and variant detection. This package was developed at NCGR, the home of collaborators, Greg May and Joann Mudge. SNP Assays. For each species, 2000 SNPs will be assayed on 384 accessions using the Illumina Bead Station. This involves silica beads that self assemble into microwells of fiber optic bundles. Each bead is covered with a specific oligonucleotide that acts as a capture sequence for each SNP marker as part of the GoldenGate assay system (template extension, PCR with dye labeled, allele specific primers, hybridization to beads, visualization). Association Mapping. Well-characterized collections of accessions are key to successful association mapping. In M. truncatula, our lines come from a characterized a collection of inbreds, analyzed previously using microsatellites. Soybean lines for SNP analysis will consist of parents used in breeding crosses, exotic sources of disease resistance, and Minnesota-adapted germplasm coming from the resistance breeding program. Decisions about the best alfalfa cultivars for analysis are still in progress. To analyze association mapping data, we will use a linear mixed model to evaluate each SNPs association with traits of interest (e.g., nodule number for symbiosis, cyst number for SCN). To account for the large number of tests, we will calculate false discovery rate and use it to adjust p values. We will also use simulation approaches to evaluate genome wide significance level. A major challenge for any association study is differentiating SNP phenotype associations due to tight linkage from spurious associations caused by population structure. To control for the effects of populations structure, we will estimate ancestry using either a model based approach or a data driven approach like principle component analysis, and then include the estimated components as fixed effects in the regression analysis.

Progress 10/01/08 to 09/30/13

Outputs
Target Audience: TARGET AUDIENCES Crop Breeders, primarily soybean breeders. Plant Pathologists, primarily scientists interested in soybean cyst nematodes. Plant Geneticists, primarily scientists working on the evolution of disease resistance genes, especially in legume species. Plant Genomicists, targeting reseachers carrying out large-scale, whole-genome sequencing projects of plant species, especially legumes. Plant Bioinformaticists, especially programmers developing software for discovery and analysis of gene involved in plant-microbe interactions. EFFORTS Research presentations to large and small target audiences Formal classroom instruction to graduate students in field of plant genomics Formal classroom instuction to undergraduates in field of biotechnology Undergraduate interns and research fellows mentored in plant genetics and bioinformatics Graduate students and post-doctoral fellows mentored in plant genetics and bioinformatics Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? A total of four graduate students (one of whom is hispanic), one post-doc, and more than 10 undergraduates (six of whom are hispanic and one a veteran) have participated in this project. The graduate students worked on research that ranged from (1) authoring software for discovery of secreted plant peptide gene families, (2) analyzing the evolution and genomic architecture of nodulation-related gene families in Medicago, and (3) developing genomic prediction models for SCN resistance in soybean. The post-doc working on the project has been leading the work on functional analysis of candidate genes involved in nodulation. The undergraduates, who primarily worked as summer research fellows, pursued projects to (1) identify and clone important genes expressed by the microbial partner of nitrogen fixation (Rhizobium), (2) develop modified strains that are easier to monitor, and (3) characterize possible functions of selected candidate genes from the association mapping work. For all the graduate, undergraduate and post-doc advisees, there are regular sessions with the project director, meeting with overall project personnel, and institution-level programs to explore and actively discuss issues in scientific professionalism. Every one of these advisees has prepared at least one (and often multiple) posters and/or oral presentations, with graduate students and post-docs traveling to national or international meetings to give talks. How have the results been disseminated to communities of interest? In addition to the 13 refereed publications related to research on this project (listed elsewhere), project leader (Young) has given 15 presentations at scientific meetings. Venues for these presentations include (partial list): International Legume Genetics and Genomics Conference lecture DuPont / Pioneer invited seminar Land of Lakes invited workshop presentation International Plant and Animal Genome Conference lecture University of Southern California department seminar Model Legume Congress lecture Carlson University department seminar University of Wisconsin-River Falls department seminar Univeristy of Minnesota department seminar Minnesota Supercomputer Institute invited lecture Sigma Xi society of Minnesota plenary lecture What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? GOAL 1 (MEDICAGO TRUNCATULA GENOMICS): In Medicago, we have collaborated with institutions worldwide to re-sequence the M. truncatula genome using “next generation” Illumina sequencing leading to the discovery of more than 5 million single nucleotide polymorphism (SNP markers). We utilized an optimized informatics pipeline for discovering these SNP markers at such a high density that we could go on to discover quantitative trait loci (QTLs) controlling agriculturally important traits through a gene mapping strategy known as “genome-wide association analysis” (GWAS). As a result, we identified dozens of candidate genes that are associated with nodulation and nitrogen fixation. Preliminary "reverse genetics" experiments involving plants where candidate genes have been knocked-out indicate that some of these candidate genes do, indeed, play a role in nodulation. We also discovered hundreds of nodule-localized defensin genes, which are genes specific to Medicago and close relatives. They play a key role in nodulation and symbiosis. To enable this work, students working on the project developed new types of bioinformatic data-mining software that efficiently discovers gene families of interest from both complete genome sequences and assemblies generated from next generation sequencing. This inventory of Medicago symbiosis, resistance and defensin genes facilitates the discovery and utilization of genes for symbiosis and disease resistance in legumes generally and in alfalfa more specifically. Using the M. truncatula genomes and SNP database as a starting point, we went on to carry out next targeted next generation sequencing of additional M. truncatula relatives, including a widely-studied genotype known as R108 as well as alfalfa (M. sativa). In addition to finding millions of SNPs between amont these (relatively) close taxa, we found that nodule-localized defensin genes are highly similar in both M. truncatula, R108 and M. sativa. Surprisingly, some families of these nodule-related genes show notable genomic differences compared to another related forage legume, red clover (Trifolium pratense). GOAL 2 (SOYBEAN MARKER-ASSISTED BREEDING AND SNP DEVELOPMENT): Project scientists have focused on soybean cyst nematode (SCN) resistance genes, including the well-known Rhg1 (on chromosome 18) as well as a newly described resistance gene on chromosome 10. This new resistance gene comes from the exotic soybean accession PI516567C and is effective against a different array of SCN races than Rhg1. This is significant because growers are finding that existing “resistant” varieties are unable to stop the growth and spread of new races of SCN. Integrating a new resistance gene with different race specificities should improve soybean grower options and profitability. We worked to optimize DNA marker-based screening of SCN resistant germplasm. Based on dozens of SNP markers near SCN resistance genes, we assayed soybean crosses and progeny in the Minnesota breeding program in terms of which alleles have been inherited, whether lines harbor new forms of resistance, and whether crossover events may have occurred in flanking regions. These SNP-based tools are fully integrated into the soybean breeding program at the University of Minnesota. Recently, our soybean molecular breeding work has been extended through the use of Goldengate 1,536 SNP arrays to develop genomic selection prediction models for quantitative SCN resistance (involving new and previously uncharacterized resistance loci) along with other agronomic and disease resistance phenotypes. Results of our association mapping and genomic selection work indicate that genomic prediction models based on these 1536 SNPs uncover loci already known for SCN resistance as well as previously uncharacterized variation only observed through analysis of the genome-wide collection of SNP markers. Our study demonstrates that association mapping can be an effective tool for identifying resistance genes of interest in diverse germplasm. Our results also indicate that improved genomic selection based on genome-wide SNP markers can enhance breeding efficiency for SCN resistance in existing soybean improvement programs. Potentially, this previously unexplored variation in SCN resistance will be valuable in breeding new and better soybean varieties.

Publications

• Type: Journal Articles Status: Published Year Published: 2012 Citation: Young ND, Bharti AK (2012) Genome-enabled insights into legume biology. Annual Review of Plant Biology 12: 193-201.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Paape T, Zhou P, Branca A, Briskine R, Young ND, Tiffin P (2012) Fine scale population recombination rates, hotspots and correlates of recombination in the Medicago truncatula genome. Genome Biology and Evolution 4: 726-737. doi: 10.1093/gbe/evs046.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Epstein B, Branca A, Mudge J, Bharti AK, Briskine R, Farmer A, Sugawara M, Young ND, Sadowsky MJ, Tiffin P (2012) Population genomics of the facultatively mutualistic bacteria Sinorhizobium meliloti and S. medicae. PLoS Genetics 8(8): e1002868. doi:10.1371/journal.pgen.1002868.
• Type: Journal Articles Status: Published Year Published: 2012 Citation: Ashfield T, Egan AN, Pfeil BE, Chen NWG, Podicheti R, Ratnaparkhe MB, Ameline-Torregrosa C, Denny R, Cannon S, Doyle JJ, Geffroy V, Roe BA, Saghai Maroof MA, Young ND, Innes RW (2012) Evolution of a complex disease resistance gene cluster in diploid Phaseolus and tetraploid Glycine. Plant Physiology 159: 344-354. doi:10.1104/pp.112.195040.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhou P, Silverstein KAT, Gao L, Walton JD, Nallu S, Guhlin J, Young ND (2013) Detecting small plant peptides using SPADA (Small Peptide Alignment Discovery Application). BMC-Bioinformatics 14: 335. doi: 10.1186/1471-2105-14-335.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yoder JB, Briskine, R, Mudge J, Farmer A, Paape T, Steel K, Weiblen GD, Bharti A, Zhou P, May GD, Young ND, Tiffin P (2013) Phylogenetic signal variation in the genomes of the genus Medicago (Fabaceae). Systematic Biology 62(3): 424-438. doi:10.1093/sysbio/syt009.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Sugawara M, Epstein B, Badgley B, Unno T, Xu L , Reese J, Gyaneshwar P, Denny R, Mudge J, Bharti AK, Farmer AD, May GD, Woodward JE, M�digue C, Vallenet D, Lajus A, Rouy Z, Martinez-Vaz B, Tiffin P, Young ND, Sadowsky MJ (2013) Comparative genomics of the core and accessory genomes of 48 Sinorhizobium strains spanning five genospecies. Genome Biology 14(2):R17. doi:10.1186/gb-2013-14-2-r17.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Paape T, Bataillon T, Zhou P, Kono T, Briskine R, Young ND, Tiffin P (2013) Selection, genome wide fitness effects, and evolutionary rates in the model legume Medicago truncatula. Molecular Ecology 22(13): 3525-3538. doi: 10.1111/mec.12329.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Bonhomme M, Andr� O, Badis Y, Ronfort J, Burgarella C, Chantret N, Prosperi J-M, Briskine R, Mudge J, Deb�ll� F, Navier H, Miteul H, Hajri A, Baranger A, Tiffin P, Dumas B, Pilet-Navel M-L, Young ND, Jacquet C (2014) High density genome-wide association mapping implicates an F-box encoding gene in Medicago truncatula resistance Aphanomyces euteiches. New Phytologist: 201(4): doi: 10.1111/nph.12611.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Stanton-Geddes J, Paape T, Epstein B, Briskine R, Yoder, J, Mudge J, Bharti AK, Farmer AD, Zhou P, Denny R, May GD, Erlandson S, Sugawara M, Sadowsky MJ, Young ND, Tiffin P (2013) Candidate genes and genetic architecture of symbiotic and agronomic traits revealed by whole-genome, sequence-based association genetics in Medicago truncatula. PLoS ONE 8(5): e65688. doi:10.1371/journal.pone.0065688.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Stanton-Geddes J, Yoder J, Briskine R, Young ND, Tiffin P (2013) Estimating heritability with whole-genome data. Methods in Ecology and Evolution: doi: 10.1111/2041-210X.12129.

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

Outputs
OUTPUTS: Research on this CRIS project examines the genomics of two key legumes, soybean (Glycine max) and the model, Medicago truncatula. The project's primary goal is characterizing the genome architecture of legume genes involved in plant-microbe interaction and extending this knowledge to develop improved crop varieties. Members of the project characterize the genomes of soybean and M. truncatula through a combination of DNA sequencing and bioinformatic data-mining. Research on M. truncatula is especially relevant in crop improvement due to its close taxonomic relationship to alfalfa (M. sativa), enabling efficient translation of discoveries to alfalfa improvement. Specific project outputs include: 1) Examining genetic variation in and around major soybean resistance genes for cyst nematode (SCN); 2) Describing global genetic architecture of disease resistance and defensin-related genes throughout legume genomes; 3) Surveying DNA sequence variation among resistance genes in Medicago and soybean, and 4) Discovering single nucleotide polymorphisms (SNPs) in Medicago as a basis for genome-wide association mapping of genes underlying symbiosis with Rhizobium bacteria and mycorrhizal fungi. The project also plays a major role in the design and implementation of "Goldengate" SNP marker platforms for both soybean and Medicago as well as development of bioinformatic pipelines for the discovery of secreted peptides that play a role in plant-microbe interactions. To communicate outputs of our research to the research community, the project maintains a fully featured and highly popular website (www.medicagohapmap.org) that receives more than a 1000 unique visits (roughly 15,000 hits) each month. PARTICIPANTS: University of Minnesota participants: Senyu Chen James Orf Mike Sadowsky Kevin Silverstein Robert Stupar Peter Tiffin Partner Organizations: Steven Cannon, ARS-Iowa State University Maria Harrison, Boyce Thompson Institute, Cornell University (New York) Maria Monteros, Noble Foundation, Ardmore, OK Michael Udvardi, Noble Foundation, Ardmore, OK Joann Mudge, National Center for Genome Resources (NCGR; New Mexico) Chris Town, J. Craig Venter Institute (JCVI, Maryland) John Innes Centre (Norwich, UK) Institut Nationale de Recherche Agronomique (INRA; Toulouse, FR) Ecole National Superieur Agronomique de Toulouse (ENSAT; Toulouse, FR) TARGET AUDIENCES: Crop Breeders, Plant Geneticists, Plant Genomicists, Plant Bioinformaticists PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
In soybean, project scientists focus on SCN resistance genes, including the well-known Rhg1 (on chromosome 18) plus a newly described gene on chromosome 10. This new resistance gene comes from soybean accession PI516567C and is effective against a different array of SCN races than Rhg1. This is significant because growers are finding that existing "resistant" varieties are unable to stop the growth and spread of SCN. Integrating a new resistance gene with different race specificities should improve soybean grower options and profitability. Project scientists continue to optimize marker-based screening of SCN resistant germplasm. Based on dozens of SNP markers near SCN resistance genes, we assay soybean crosses and progeny in the Minnesota breeding program in terms of which alleles have been inherited, whether lines harbor new forms of resistance, and whether crossover events may have occurred in flanking regions. These SNP-based tools are fully integrated into the soybean breeding program at the University of Minnesota. Recently, our work has been extended through the use of Goldengate 1,536 SNP arrays to develop genomic selection prediction models for quantitative SCN resistance (involving new and previously uncharacterized resistance loci) along with other agronomic and disease resistance phenotypes. In Medicago, we have collaborated with institutions worldwide to re-sequencing of the M. truncatula genome using "next generation" Illumina sequencing technology leading to the discovery of more than 5 million SNP markers. We utilize an optimized informatics pipeline for discovering these SNP markers at such a high density that we can go onto discover quantitative trait loci (QTLs) controlling agriculturally important traits through a process known as "genome-wide association mapping." As a result, we have identified dozens of candidate genes that seem to be associated with contemporary evolution of nodulation and nitrogen fixation. We have also discovered hundreds of nodule-localized defensin genes, which are genes specific to Medicago and close relatives that play a key role in nodulation and symbiosis. This inventory of Medicago symbiosis, resistance and defensin genes facilitates the discovery and utilization of genes for symbiosis and disease resistance in legumes generally and in alfalfa more specifically.

Publications

• Ashfield T, Egan AN, Pfeil BE, Chen NWG, Podicheti R, Ratnaparkhe MB, Ameline-Torregrosa C, Denny R, Cannon S, Doyle JJ, Geffroy V, Roe BA, Saghai Maroof MA, Young ND, Innes RW (2012) The impact of polyploidy on the evolution of a complex NB-LRR resistance gene cluster in soybean. Plant Physiology 159: 344-354.
• Epstein B, Branca A, Mudge J, Bharti AK, Briskine R, Farmer A, Sugawara M, Young ND, Sadowsky MJ, Tiffin P (2012) Population genomics of the facultatively mutualistic bacteria Sinorhizobium meliloti and S. medicae. PLoS Genetics 8(8): e1002868. doi:10.1371/journal.pgen.1002868.
• Paape T, Zhou P, Branca A, Briskine R, Young ND, Tiffin P (2012) Fine scale population recombination rates, hotspots and correlates of recombination in the Medicago truncatula genome. Genome Biology and Evolution 4: 726-737.
• Young ND, Bharti AK (2012) Genome-enabled insights into legume biology. Annual Review of Plant Biology 12: 193-201.

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

Outputs
OUTPUTS: Research on this CRIS project targets the comparative genomics of legumes, primarily soybean (Glycine max) and model legume, Medicago truncatula. The project's primary goal is characterizing the evolution of legume genes involved in symbiosis and disease resistance and then extending this knowledge to develop improved crop varieties. Members of the project characterize the genomes of soybean and M. truncatula through a combination of DNA sequencing and bioinformatic data-mining. Though Medicago is best known as a model system, it is especially relevant in crop improvement due to its close taxonomic relationship to alfalfa (M. sativa). This enables efficient translation of Medicago genomic discoveries to alfalfa improvement. Specific project outputs include: 1) Describing genetic variation in and around major resistance genes for soybean cyst nematode (SCN) in soybean, 2) Describing the global genetic architecture of disease resistance and defensin-related genes throughout the Medicago genome, 3) Surveying genetic variation among resistance genes in Medicago and soybean, and 4) Using next-generation sequencing techniques to discover single nucleotide polymorphisms (SNPs) in Medicago as a basis for genome-wide association mapping and discovery of genes underlying symbiosis with Rhizobium bacteria and mycorrhizal fungi. To communicate outputs of our research to the broader research community, the project maintains a fully featured and highly popular website (www.medicagohapmap.org) that receives more than a 1000 unique visits (roughly 15,000 hits) per month. According to the 2009 NSF plant website survey, this site is one of the most frequent destinations for plant genomics researchers and hit frequencies have increased still further during this reporting period. PARTICIPANTS: Participants: University of Minnesota participants: Senyu Chen James Orf Mike Sadowsky Kevin Silverstein Peter Tiffin Partner Organizations: Steven Cannon, ARS-Iowa State University Maria Harrison, Boyce Thompson Institute, Cornell University (New York) Joann Mudge, National Center for Genome Resources (NCGR; New Mexico) Chris Town, J. Craig Venter Institute (JCVI, Maryland) John Innes Centre (Norwich, UK) Institut Nationale de Recherche Agronomique (INRA; Toulouse, FR) Ecole National Superieur Agronomique de Toulouse (ENSAT; Toulouse, FR) TARGET AUDIENCES: ** Crop Breeders ** Plant Geneticists ** Plant Genomicists ** Plant Bioinformaticists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In soybean, project scientists focus on SCN resistance genes, including the well-known Rhg1 (on chromosome 18) plus a newly described gene on chromosome 10. This new resistance gene comes from soybean accession PI516567C and seems to be effective against a different array of SCN races than Rhg1. This is significant because growers are finding that existing "resistant" varieties are unable to stop the growth and spread of SCN. Integrating a new resistance gene with different race specificities should improve soybean grower options and profitability. Project scientists continue to optimize marker-based screening of SCN resistant material. SNP markers trace ancestry, genetic recombination, and select favorable lines based on sequence polymorphisms surrounding Rhg1, the new resistance gene on chromosome 10, and another important resistance gene, Rhg4. Based on dozens of SNP markers near SCN resistance genes, it is now possible to assay soybean crosses and progeny in terms of which alleles have been inherited, whether lines harbor new forms of resistance, and whether crossover events may have occurred in flanking regions. These SNP-based tools are fully integrated into the soybean breeding program at the University of Minnesota. In Medicago, we have collaborated with institutions worldwide to complete the genome sequence of this model species. In the process, we described the entire gene complement and identified more than 700 NBS-LRR type resistance genes, the highest number for any sequenced dicot so far. NBS-LRR genes in Medicago exhibit a remarkably high degree of gene clustering, numerous translocations plus large numbers of novel gene rearrangements. We also discovered hundreds of nodule-localized defensin genes. These genes are specific to Medicago and close relatives. Nonetheless, they play a key role in nodulation and nitrogen-fixation. This inventory of Medicago resistance and defensin genes facilitates the discovery and utilization of genes for symbiosis and disease resistance in legume generally and in alfalfa specifically. Sequencing M. truncatula also revealed a key genome duplication event approximately 58 million years ago. Analysis of sequence data indicates this event was important in the evolution of symbiosis and nitrogen-fixation. Some of the critical genes responsible for recognition of symbiotic partners came about as a result of the genome duplication event through a process known as "genetic sub-functionalization". Subsequent re-sequencing of the M. truncatula genome using "next generation" Illumina sequencing technology has enabled us to discover more than 5 million SNP markers. We utilize an optimized informatic pipeline for discovering these markers so resequencing is easily extended to hundreds of different Medicago lines as well as cultivated alfalfa. SNP markers at such a high density is now providing a framework for discovery of quantitative trait loci (QTLs) controlling agriculturally important traits in a process known as "genome-wide association mapping."

Publications

• Branca A, Paape T, Zhou P, Briskine R, Farmer AD, Mudge J, Bharti AK, Woodward JE, May GD, Gentzbittel L, Ben C, Denny R, Sadowsky, MJ, Ronfort J, Bataillon T, Young ND, Tiffin P (2011) Whole-genome nucleotide diversity, recombination, and linkage-disequilibrium in the model legume Medicago truncatula. Proc. Natl. Acad. Sci. U.S.A. doi:10.1073/pnas.1104032108.
• Young N, Debelle F, Oldroyd G, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KFX, Gouzy J, Schoof H, et al. (2011) The Medicago genome provides insight into the evolution of rhizobial symbioses. Nature. DOI 10.1038/nature10625.

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

Outputs
OUTPUTS: Research on this CRIS project focuses on the comparative genomics of legumes, primarily soybean (Glycine max) and the model legume, Medicago truncatula. The project's specific goal is characterizing the evolution of legume genes and genomes, primarily genes involved in symbiosis and disease resistance, and then extending this knowledge to develop improved varieties for crop improvement. We are characterizing the genomes of soybean and Medicago truncatula through a combination of DNA sequencing and bioinformatic data-mining. Though Medicago is a model system, it is especially relevant due to its close taxonomic relationship to alfalfa (M. sativa). This enables efficient translation of Medicago genomic discoveries to alfalfa improvement. Specific project outputs include: 1) Describing genetic variation in and around major resistance genes for soybean cyst nematode (SCN) in soybean, 2) Describing the global genetic architecture of disease resistance genes throughout the Medicago genome, 3) Surveying genetic variation among resistance genes in Medicago and soybean, and 4) Using next-generation sequencing techniques to discover single nucleotide polymorphisms (SNPs) in Medicago as a basis for genome-wide association mapping, genome sequencing in alfalfa, and discovery of genes underlying symbiosis with Rhizobium bacteria and mycorrhizal fungi. To communicate outputs of our research to the broader research community, we maintain a fully featured website (www.medicago.org) that receives more than a 1000 unique visits (roughly 15,000 hits) per month. Our site is one of the most frequent destinations for plant genomics researchers according to a 2009 National Science Foundation survey. PARTICIPANTS: University of Minnesota participants: James Orf Mike Sadowsky Peter Tiffin Partner Organizations: Steven Cannon, ARS-Iowa State University Maria Harrison, Boyce Thompson Institute, Cornell University (New York) Joann Mudge, National Center for Genome Resources (New Mexico) Sergey Nuzhdin, University of Southern California TARGET AUDIENCES: Crop Breeders, Plant Geneticists, Plant Genomicists, Plant Bioinformaticists PROJECT MODIFICATIONS: None

Impacts
In soybean, we focus on disease resistance genes against SCN, primarily the best characterized examples, Rhg1 and Rhg4. In parallel, we also search for novel sources of resistance genes by scanning the genomes of other potential SCN resistant lines. Our work begins with the identification and characterization of SNP markers that can be used to trace ancestry, genetic recombination, and select favorable breeding lines based on sequence polymorphisms surrounding Rhg1 and Rhg4 and that can be used to confirm the existence of novel resistance genes. Based on dozens of SNP markers we've examined in the regions around SCN resistance genes, it is now possible to assay soybean crosses and progeny in terms of which alleles have been inherited, whether newly tested soybean lines harbor new forms of SCN resistance, and whether crossover events may have occurred in or around SCN resistance genes. The ability to ask these questions through the use of SNPs is now fully integrated into the soybean breeding program at the University of Minnesota. In Medicago, we have identified nearly 700 NBS-LRR type resistance genes, the highest number for any sequenced dicot so far. Certain evolutionary features are pronounced in Medicago, including a remarkably high degree of gene clustering, numerous translocations from gene clusters to other parts of the genome, a small number of evolutionarily more stable NBS-LRRs, plus large numbers of novel gene rearrangements. Of notable significance is the observation that many NBS-LRR resistance genes in Medicago are significantly expressed in nodules, the organ that is responsible for symbiotic nitrogen fixation. This inventory of Medicago resistance genes and knowledge about their diversity and expression facilitates the discovery and utilization of genes for disease resistance in alfalfa. Resequencing of the Medicago truncatula genome through the use of "next generation" Illumina sequencing technology has enabled us to discover more than 5 million SNP markers in this model legume species. We utilize an optimized informatic pipeline for discovering these markers so resequencing is easily extended to hundreds of different Medicago lines as well as cultivated alfalfa. SNP markers at such a high density allow us to define the haplotype blocks that comprise present day Medicago germplasm, providing a framework for discovery of quantitative trait loci (QTLs) controlling agriculturally important traits, especially symbiosis, nodulation and nitrogen fixation.

Publications

• None in 2010

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

Outputs
OUTPUTS: Research on this CRIS project focuses on the comparative genomics of legumes, primarily soybean (Glycine max) and the model legume, Medicago truncatula. Our specific goal is characterizing the evolution of legume genes and genomes, especially genes involved in plant-microbe interactions, and then extending this knowledge to the development of improved germplasm for use in crop improvement. We are characterizing the genomes of soybean and Medicago truncatula through a combination of DNA sequencing and bioinformatic data-mining. Research in Medicago is especially relevant because of its close taxonomic relationship to alfalfa (M. sativa), leading to the possibility of quickly translating Medicago genomic discoveries to alfalfa improvement. In the process we are: 1) Describing the genetic architecture of disease resistance genes throughout the Medicago genome, 2) Comparing the structure and organization of major resistance gene clusters between Medicago and soybean, 3) Determining sequence variation among resistance genes in Medicago and soybean, and 4) Using next-generation sequencing techniques to discover single nucleotide polymorphisms (SNPs) in Medicago as a basis for genome-wide association mapping, genome sequencing in alfalfa, and investigations into the genetic basis of symbiosis. To communicate the outputs of our research to broader research community, we maintain a fully featured website (www.medicago.org) that receives more than a 1000 unique visits (roughly 15,000 hits) per month. This is one of the highest number of regular visits for any plant genomics website in the US according to a recent National Science Foundation survey. PARTICIPANTS: University of Minnesota participants: James Orf, Mike Sadowsky, Peter Tiffin. Partner Organizations: Steven Cannon, ARS-Iowa State University; Maria Harrison, Boyce Thompson Institute, Cornell University; Joann Mudge, National Center for Genome Resources; Sergey Nuzhdin, University of Southern California TARGET AUDIENCES: Crop Breeders, Plant Geneticists, Plant Genomicists, Plant Bioinformaticists PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
In soybean, we focus on the disease resistance genes, Rpg1 (resistance to bacterial blight) and Rhg1 (resistance to soybean cyst nematode). We find that the Rpg1 resistance gene cluster has undergone dramatic expansions and rearrangements compared with duplicated segments of the soybean genome, including partitioning of different resistance gene sub-families between the duplicated segments. Potentially, the partitioning of different resistance genes between duplicate resistance gene clusters provides plants with expanded capacity to respond to novel pathogenes. Overall, the organization of genes near Rpg1 is conserved among different soybean cultivars, although there is substantial variation in the precise number of resistance genes located in each cluster. Again, this high level of variation in resistance gene clusters probably plays an important role in disease resistance flexibility for the plant. In the Rhg1 genome region, our focus is on the identification and characterization of SNP markers that can be used to trace ancestry, genetic recombination, and select favorable breeding lines based on sequence polymorphisms surounding Rhg1. Based on dozens of SNP markers we've examined in the region around Rhg1, it is now possible to assay soybean crosses and progeny in terms of which Rhg1 allele has been inherited and whether any nearby cross-over events may have occurred. The ability to ask these questions through the use of SNPs is now being integrated into the soybean breeding program at the University of Minnesota. In Medicago, we identified more than 600 NBS-LRR type resistance genes, the highest number for any sequenced dicot so far. Certain evolutionary features are pronounced in Medicago, including a high degree of gene clustering, numerous translocations from gene clusters to other parts of the genome, a small number of evolutionarily more stable NBS-LRRs, plus numerous examples of novel gene rearrangements. This inventory of Medicago resistance genes facilitates the discovery and utilization of genes for disease resistance in alfalfa. Resequencing of the Medicago truncatula genome through the use of "next generation" sequencing technology has enabled us to discovery more than 3 million SNP markers in this model legume species. We have recently optimized the informatic pipeline for discovering these markers so resequencing can be extended to hundreds of different Medicago lines (including cultivated alfalfa). SNP markers at such high density allow us to define the haplotype blocks that comprise present day Medicago germplasm, providing a framework for discovery of quantitative trait loci (QTLs) controlling agriculturally important traits, especially symbiosis, nodulation and nitrogen fixation.

Publications

• Young, N.D., Udvardi, M. (2009) Translating Medicago truncatula genomics to crop legumes. Current Opinions in Plant Biology. 12: 193-201.

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

Outputs
OUTPUTS: Research on this CRIS project focuses on the comparative genomics of legumes. Our primary goal is characterizing the evolution of legume genes and genomes, especially disease resistance genes, and extending this knowledge to the development of improved germplasm for use in breeding. Specifically, we are characterizing the genomes of soybean and the model legume, Medicago truncatula, through a combination of DNA sequencing and bioinformatic data-mining. Research in Medicago is especially promising because of its close taxonomic relationship to alfalfa (M. sativa), leading to the possibility of quickly translating Medicago genomic discoveries to alfalfa improvement. In the process, we are: 1) Describing the genetic architecture of disease resistance genes throughout the Medicago genome, 2) Comparing the structure and organization of major resistance gene clusters between Medicago and soybean, 3) Determining sequence variation among resistance genes in Medicago and soybean, 4) Characterizing single nucleotide polymorphisms (SNPs) that distinguish sequence variation among resistance genes in soybean, and 5) Using next-generation sequencing techniques to discover SNPs in Medicago as a basis for genome-wide association mapping and genome sequence development in alfalfa. PARTICIPANTS: University of Minnesota participants: James Orf Mike Sadowsky Peter Tiffin Partner Organizations: Roger Innes, Indiana University Steven Cannon, ARS-Iowa State University TARGET AUDIENCES: ** Crop Breeders ** Plant Geneticists ** Plant Genomicists ** Plant Bioinformaticists PROJECT MODIFICATIONS: None

Impacts
In soybean, we have studied two important disease resistance genes: Rpg1 (resistance to bacterial blight) and Rhg1 (resistance to soybean cyst nematode). We have found that the Rpg1 resistance gene cluster has undergone dramatic expansions and rearrangements compared with duplicated segments of the soybean genome, including partitioning of different resistance gene sub-families between the duplicated segments. Overall, the organization of genes near Rpg1 is conserved among different soybean cultivars, although there is substantial variation in the precise number of resistance genes located in each cluster. We also found that the retention rate of gene duplicates in soybean is much higher than that reported for corn. These results help to understand the evolutionary trends that drive diversification of disease resistance genes in soybean and crop plants generally. In the Rhg1 genome region, our focus is on the identification and characterization of single nucleotide polymorphism (SNP) markers that can be used to trace ancestry and genetic recombination in and around Rhg1. By characterizing 35 SNP markers located in the genome regions surrounding Rhg1, it is now possible to assay soybean crosses and progeny populations in terms of which form of the Rhg1 gene (allele) has been inherited and whether any nearby cross-over events have occurred. The ability to ask these questions through the use of SNPs is leading to more efficient breeding of cyst nematode resistant cultivars. In Medicago, we identified 333 members of the NBS-LRR superfamily of disease resistance genes, a number more than two times greater than in the model, Arabidopsis thaliana. Certain evolutionary features are pronounced in Medicago, including a high degree of gene clustering, numerous translocations from gene clusters to other parts of the genome, a small number of more stable NBS-LRRs, and numerous examples of novel gene rearrangements. This inventory of Medicago resistance genes facilitates the discovery and utilization of genes for disease resistance in alfalfa.

Publications

• Ameline-Torregrosa, C., Cazaux, M., Danesh, D., Chardon, F., Cannon, S.B., Esquerre-Tugaye, M.T, Dumas. B., Young, N.D., Samac, D.A., Huguet. T., Jacquet, C. (2008) Genetic dissection of resistance to anthracnose and powdery mildew in Medicago truncatula. Molecular Plant-Microbe Interaction 21: 61-69.
• Ameline-Torregrosa, C., Wang, B.-B., Denny, R.L., OBleness, M., Despande, S., Zhu, H., Roe, B., Young, N.D., Cannon, S.B. (2008) Identification and characterization of NBS-LRR encoded genes in the model plant Medicago truncatula. Plant Physiology 146: 5-21.
• Hyten, D.L., Song, Q., Choi, I.-Y., Yoon. M.-S., Specht. J.E., Matukumalli, L.K,, Nelson. R.L., Shoemaker, R.C., Young, N.D., Cregan, P.B. (2008) High-throughput genotyping with the GoldenGate assay in the highly complex genome of soybean. Theoretical and Applied Genetics, 116: 945-952.
• Innes, R.W., Ameline-Torresgrosa, C., Ashfield, T., Cannon, C., Cannon, S.B., Chacko, B., Chen, N.W.G., Couloux, A., Dalwani, A., Denny, R., Deshpande, S., Egan, A..N., Glover, N., Hans, C.S., Howell, S., Ilut, D., Jackson, S., Lai, H., Mammadov, J., del Campo, S., Metcalk, M. Nguyen, A., OBleness, M., Pfeil, B.E., Podicheti, R., Ratnaparkhe, M.B., Samain, S., Snaders, I. Seguren, B., Sevignac, M., Sherman-Broyles, S., Thareau, V., Tucker, D.M., Walling, J., Wawrzynski, A., Yi, J., Doyle, J.J., Geffroy, V., Roe, B.A., Maroof, S., Young, N.D. (2008) Differential accumulation of retroelements and diversification of NB-LRR disease resistance genes in duplicated regions following polyploidy in the ancestor of soybean. Plant Physiology 148(4): 1740-1759.
• Wawrzynski A, Ashfield T, Chen NW, Mammadov J, Nguyen A, Podicheti R, Cannon SB, Thareau V, Ameline-Torregrosa C, Cannon E, Chacko B, Couloux A, Dalwani A, Denny R, Deshpande S, Egan AN, Glover N, Howell S, Ilut D, Lai H, Del Campo SM, Metcalf M, O'Bleness M, Pfeil BE, Ratnaparkhe MB, Samain S, Sanders I, Segurens B, Sevignac M, Sherman-Broyles S, Tucker DM, Yi J, Doyle JJ, Geffroy V, Roe BA, Maroof MA, Young ND, Innes RW. (2008) Replication of non-autonomous retroelements in soybean appears to be both recent and common. Plant Physiology 148(4): 1760-1771.

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

Outputs
OUTPUTS: Research on this CRIS project focuses on the comparative genomics of legumes. The primary goal is characterizing the evolution of disease resistance genes and extending this knowledge in the development of resistant germplasm. More specifically, we are characterizing the genomes of soybean, wild soybean relatives, and the model legume, Medicago truncatula, through a combination of DNA sequencing and bioinformatic data-mining. In the process, we are: 1) describing the genetic architecture of disease resistance genes throughout the Medicago genome, 2) comparing the structure and organization of several major resistance gene clusters between Medicago and soybean, 3) determining sequence variation among and between resistance genes in soybean, and 4) characterizing single nucleotide polymorphisms (SNPs) that distinguish sequence variation among resistance genes in soybean. PARTICIPANTS: YOUNG LAB AT UNIV. OF MINNESOTA: R. Denny; B.B. Wang; X. Wang; PARTNERS & COLLABORATORS: S.B. Cannon, USDA-ARS/Department of Agronomy, Iowa State Univ.; C.P. Cregan, USDA-ARS/Beltsville, MD; J.J. Doyle, Cornell Univ.; R. Innes, Indiana Univ.; S. Maroof, Virginia Tech Univ.; J.H. Orf, Department of Agronomy and Plant Genetics, Univ. of Minnesota; B.A. Roe, Department of Biochemistry, Univ. of Oklahoma; D. Samac, USDA-ARS/Department of Plant Pathology, Univ. of Minnesota; C.D. Town, J. Craig Venter Institute TARGET AUDIENCES: Basic researchers in the field of plant genomics. Basic researchers in the field of plant disease resistance biology. Plant breeders developing new crop varieties.

Impacts
In the process of studying legume resistance genes, we have concentrated on a large gene cluster on soybean chromosome "F" where multiple resistance specificities have been mapped, included a cloned resistance gene to bacterial blight (Rpg1). We have found that the overall organization of genes in the Rpg1 region is conserved among different soybean cultivars, although there is substantial variation in the exact number of resistance genes located there. We examined the sequence of two resistance genes in detail in dozens of different soybean cultivars and found that significant diversity exists. Surprisingly, some of this sequence variation has arisen in just the last 100 years (the period of modern soybean breeding). Potentially, this variation is due to high levels of gene conversion, but more research is needed. This comparative genomic analysis is providing insight into how legumes generate disease resistance diversity, as well as the mechanisms plants use to defend themselves and recognize novel pathogens.

Publications

• Choi, I.-Y., Hyten, D.L., Specht, J.E., Matukumalli, L.K., Song, Q., Quigley, C.V., Lee, M.S., Chase, K., Lark, K.G., Reiter, R.S., Yoon, M.-S., Hwang, E.-Y., Yi, S.-I., Young, N.D., Shoemaker, R.C., van Tassell, C.P., Cregan, P.B. 2007. A soybean transcript map: Discovery and mapping of single nucleotide polymorphisms in soybean genes. Genetics 176: 685-696.
• Febrer, M., Cheung, F., Town, C., Cannon, S., Young, N.D., Abberton, M., Jenkins, G., Milbourne, D. 2007. Construction, characterization and preliminary BAC-end sequencing analysis of a bacterial artificial chromosome library of white clover (Trifolium repens L.). Genome 50: 412-421.
• Foster-Hartnett, D., Danesh, D., Penuela, S., Sharopova, N., Endre G., VandenBosch, K., Young, N.D., Samac, D. 2007. Cytological and molecular responses of Medicago truncatula to Erysiphe pisi. Molecular Plant Pathology 8: 307-319.
• Hyten. D.L., Choi, I.Y., Song, Q., Yoon, M.S., Specht J.E., Nelson, R.L., Carter, Jr., T.C., Chase, K., Young, N., Lark. G., Shoemaker, R.C., Cregan, P.B. 2007. An assessment of genome-wide linkage disequilibrium in soybean. Plant and Animal Genome Meeting XV, San Diego CA, January, 2007.
• Wang, B.B., Cannon, S.B., Sterck, L., Wang, S., Roe B.A., Town, C.D., Van de Peer, Y., Young, N.D. 2007. Synteny blocks are mostly small and fragmented in comparisons between Medicago and other sequenced angiosperms. Plant and Animal Genome Meeting XV, San Diego CA, January, 2007.
• Young, N.D., Roe, B.A., Town, C.D. 2007. The genome sequence of Medicago truncatula. Model Legume Congress, Tunis, Tunisia, March 2007.
• Young, N.D., Roe, B.A., Town, C.D. 2007. Insights into legume genomes from the sequence of Medicago truncatula. Integrating Legume Biology for Sustainable Agriculture Conference, Lisbon, Portugal, November, 2007.
• Young, N.D., Town, C.D., Roe, B.A. 2007. Finishing the sequence of euchromatin in the model legume, Medicago truncatula. Plant and Animal Genome Meeting XV, San Diego CA, January, 2007.

Progress 01/01/06 to 12/31/06

Outputs
Research in the lab focuses on the comparative genomics of legumes, emphasizing the evolution of disease resistance genes. We are characterizing the genomes of soybean, wild soybean relatives, and the model legume, Medicago truncatula, using a combination of DNA sequencing and bioinformatic data-mining. In the process, we have characterized large clusters of disease resistance genes and discovered genome regions with the same ensemble of genes in the same order, a phenomenon known as "microsynteny." We have isolated and characterized the disease resistance genes from soybean and Medicago and characterized their molecular diversity and evolutionary history. This information provides insight into how legumes generate disease resistance diversity, as well as the mechanisms plants use to defend themselves against pathogens. One genome regions of special interest is a large resistance gene cluster on soybean chromosome "F" where multiple resistance specificities have been mapped, included a cloned resistance gene to bacterial blight (Rpg1). We have found that the organization of genes in the Rpg1 region is generally conserved among different soybean cultivars, although there is substantial variation in the exact number of resistance genes located there. Potentially, this variation is due to differences in retrotransponsons located nearby. In terms of application, our research helps to provide breeders with more powerful tools for developing improved varieties, especially improved DNA markers for selection.

Impacts
Better understanding of disease resistance in soybean and other legumes has the potential to increase productivity and sustainability of soybean cultivation. Enhanced genetic disease resistance may lessen the need for application of chemicals. Understanding diversity among resistance genes provides a basis for developing more durable forms of genetic resistance.

Publications

• Walling, J.G., Shoemaker, R.C., Young, N.D., Mudge, J., Jackson, S.A. (2006) Chromosome level homeology in paleopolyploid soybean (Glycine max) revealed through integration of genetic and chromosome maps. Genetics 172: 1893-1900.
• Young, N.D., Shoemaker, R.C. (2006) Model legumes: Exploring the structure, function and evolution of legume genomes. Current Opinion in Plant Biology 9: 95-98.

Progress 01/01/05 to 12/31/05

Outputs
Research in the lab focuses on the comparative genomics of plants, emphasizing disease resistance genes of legumes. We are characterizing the genomes of soybean and a model legume known as Medicago truncatula, through a combination of DNA sequencing and bioinformatics. In the process, we have characterized large clusters of disease resistance genes and discovered genome regions that contain the same array of genes in the same order in both species, a phenomenon known as "microsynteny." Using powerful genomic tools, we are now isolating legume disease resistance genes and studying their molecular diversity and evolutionary history. This information will be useful in basic research, providing insights into how legumes recognize different pathogens as well as the genetic mechanisms that plants use to defend themselves against diseases. In applied research, our research will also provide breeders with more powerful tools for developing improved varieties. Specifically, our research focuses on the comparative genomics of two important chromosomal regions, one associated with resistance to soybean cyst nematode (rhg1), and a second genome region that contains resistance genes to soybean bacterial blight (Rpg1). We have found that the organization of genes in the rhg1 region is especially well-conserved between soybean, Medicago, and even the non-legume, Arabidopsis. By contrast, the Rpg1 region displays numerous rearrangments among these species. Current research focuses on the functional and evolutionary significance of the rearrangments around Rpg-1.

Impacts
Better understanding of disease resistance in soybean and other legumes has the potential to significantly increase productivity of soybean cultivation. Moreover, enhanced genetic disease resistance may lessen the need for application of chemicals.

Publications

• Cannon, S.B., Crow, J.A., Heuer, M.L., Wang, X., Cannon, E.K.S., Dwan, C., Lamblin, A.F., Vasdewani, J., Mudge, J., Cook, A., Cheung, F., Kenton, S., Kunau, T.M., Brown, D., Kim, D.J., Cook, D.R., Roe, B.A., Town, C.D., Young, N.D., Retzel, E.F. (2005) Databases and information integration for the Medicago truncatula genome and transcriptome. Plant Physiology 138: 38-46.
• Gepts, P., Beavis, W., Brummer, E.C., Shoemaker, R.C., Stalker, H.T., Weeden, N.F., Young, N.D. (2005) Legumes as a model plant family: genomics for food and feed. Report of the Cross-legume Advances Through Genomics (CATG) Conference. Plant Physiology, 137: 1228-1235.
• Lin, J.-Y., Jacobus, B. H., SanMiguel, P., Walling, J.G., Yuan, Y., Shoemaker, R.C., Young, N.D., Jackson, S.A. (2005) Centric regions of soybean (Glycine max L. Merr.) chromosomes consist of retroelements and tandemly repeated DNA and are structurally and evolutionarily labile. Genetics 170: 1221-1230.
• Mudge, J., Cannon. S.B., Kalo, P., Oldroyd. G.E.D., Roe, B.A., Town. C.D., Young. N.D. (2005) Highly syntenic regions in the genomes of soybean, Medicago truncatula, and Arabidopsis thaliana. BMC Plant Biology 5:15.
• Young, N.D., Cannon, S.B., Sato, S., Kim, D.J., Cook, D.R., Town, C.D., Roe, B.A., Tabata, S. (2005) Sequencing the genespaces of Medicago truncatula and Lotus japonicus. Plant Physiology 137: 1174-1181.

Progress 01/01/04 to 12/31/04

Outputs
Research in the lab focuses on the comparative genomics of plants, with special emphasis on the disease resistance genes of legumes. We are characterizing the genomes of soybean and a model legume known as Medicago truncatula, primarily through DNA sequencing and bioinformatics. In the process, we have discovered several regions of the genome that show remarkably similar organization in both species, a phenomenon known as "microsynteny." Using powerful genomic tools, we are now isolating large families of disease resistance genes from legumes and studying their molecular diversity and evolutionary history. This information will be useful in basic research, revealing the genetic mechanisms that plants use to defend themselves against diseases, and in also applied research, by providing breeders with better tools for developing improved varieties. Specifically, our research focuses on the comparative genomics of two important chromosomal regions, one associated with resistance to soybean cyst nematode (rhg1), the other with resistance to soybean bacterial blight (Rpg1). The organization of genes in the rhg1 region is highly conserved between soybean, Medicago, and even the non-legume, Arabidopsis. By contrast, the Rpg1 region displays numerous rearrangments among these species. Current research focuses on the functional and evolutionary significance of the highly conserved genome region surrounding rhg1 and the very different genome organization surrounding Rpg1.

Impacts
Better understanding of disease resistance and seed protein content in soybean and other legumes has the potential to significantly increase productivity of soybean cultivation. Moreover, enhanced genetic disease resistance may lessen the need for application of chemicals.

Publications

• Cannon, S.B., Mitra, A., Baumgarten, A., Young, N.D., May, G. (2004) The roles of segmental and tandem gene duplication in evolution of large gene families in Arabidopsis thaliana BMC Plant Biology 4:10 (http://www.biomedcentral.com/1471-2229/4/10)
• Choi, H.K, Mun, J.H., Kim, D.J., Zhu, H., Baek, J.M., Mudge, J., Roe, B., Ellis, T.H.N., Doyle, J., Kiss, G.B., Young, N.D., Cook, D. R. (2004) Estimating genome conservation between crop and model legume species. Proceedings National Academy Sciences USA 101:15289-15294.
• Li, Y., Chen, S.Y., Young, N.D. (2004) Effect of the rhg1 gene on penetration, development and reproduction of Heterodera glycines race 3. Nematology 6: 727-734.
• Mudge, J., Huihuang, Y., Denny, R.L., Howe, D.K., Danesh, D., Marek, L.F., Retzel, E., Shoemaker, R.C., Young, N.D. (2004) Soybean BAC contigs anchored with RFLPs: insights into genome duplication and gene clustering. Genome 47:361-372.
• Pagel, J., Walling, J.G., Young, N.D., Shoemaker, R.C., Jackson, S.A. (2004) Segmental duplications within the Glycine max genome revealed by fluorescence in situ hybridization of bacterial artificial chromosomes. Genome 47:764-768.
• Yan, H., Mudge, J., Kim, D.J., Shoemaker, R.C., Cook, D.R., Young, N.D. (2004) Comparative physical mapping reveals features of microsynteny between the genomes of Glycine max and Medicago truncatula. Genome 47:141-155.

Progress 01/01/03 to 12/31/03

Outputs
My research focuses on genome evolution of legumes, especially soybean and the model system, Medicago truncatula. Through a combination of DNA sequencing, cross-hybridization experiments, and physical mapping, my colleagues and I are examining genome regions related to two important traits: disease resistance and seed protein content. We have discovered tentative homologous (corresponding) regions for both of the traits in Medicago as a guide to genome organization and gene cloning in soybean. Specifically, we have targeted a major cluster of disease resistance genes in soybean (on chromosome F) and identified the homologous regions in Medicago. Likewise, we have targeted a specific genome region in soybean (on chromosome I) associated with high protein content and identified the corresponding regions in Medicago. Because Medicago is now the target of an international genome sequencing effort, identifying these homologous genome regions will help to reveal important properties of legume evolution regarding disease resistance and protein content. Moreover, we can begin to test candidate genes, originally found in Medicago, to see if the soybean version of the genes are responsible for the trait(s) of interest.

Impacts
Better understanding of disease resistance and seed protein content in soybean and other legumes has the potential to significantly increase productivity of soybean cultivation. Moreover, enhanced genetic disease resistance may lessen the need for application of chemicals.

Publications

• Ashfield, T., Bocian, A., Held, D., Henk, A.D., Marek, L.F., Danesh, D., Penuela, S., Meksem, K., Lightfoot, D.A., Young, N.D., Shoemaker, R.C., Innes, R.W. (2003) Genetic and physical mapping of the soybean Rpg1-b disease resistance gene reveals a complex locus containing several tightly linked families of NBS-LRR-genes. Molecular Plant-Microbe Interactions 16:817-26.
• Cannon, S.B., McCombie, W.R., Sato, S., Tabata, S., Denny, R., Palmer, L., Katari, M., Young, N.D., Stacey, G. (2003) Evolution and microsynteny of the apyrase gene family in three legume genomes. Molecular Genetics and Genomics 270: 347-361.
• Yan, H., Mudge, J., Kim, D.J., Shoemaker, R.C., Cook, D.R., Young, N.D. (2003) Estimates of conserved microsynteny among the genomes of Glycine max, Medicago truncatula and Arabidopsis thaliana. Theoretical and Applied Genetics 106: 1256-1265.
• Young, N.D., Mudge, J., Ellis, T.H.N. (2003) Legume genomes: more than peas in a pod. Current Opinion in Plant Biology 6: 199-204.
• Zhu, Y.L., Song, Q.J., Hyten, D.L., VanTassell, C.P., Matukumalli, L.K., Grimm, D.R., Hyatt, S.M., Fickus, E.W., Young, N.D., Cregan, P.B. (2003) Single nucleotide polymorphisms (SNPs) in soybean. Genetics 163: 1123-1134.

Progress 01/01/02 to 12/31/02

Outputs
MARKER-ASSISTED SELECTION FOR SOYBEAN CYST NEMATODE (SCN) RESISTANCE. One of the most immediate applications of our genomic analysis of SCN resistance has been the development of DNA markers suitable for use in marker-assisted selection. As a result of this work, several Minnesota-adapted genotypes have been tested as part of regional variety trials. These lines are part of our continuing effort to introduce new sources of SCN resistance into short maturity group soybeans. Potentially, the use of different donor genotypes can lead to greater durability of resistance. During this period, intermediate and advanced lines have been screened, primarily on the basis of the DNA markers we developed. GENOMIC ANALYSIS OF RHG1. We have completed our analysis of the soybean genome region surrounding the rhg1 SCN resistance locus. Twenty-eight large insert clones in eight distinct groups were isolated in the region around rhg1. The ends of all these clones were sequenced to estimate proportions in different sequence categories, compare similarities between soybean genome duplicates, and examine conservation with Medicago and Arabidopsis. Fingerprint, sequence, and cross-hybridization comparisons between duplicated regions revealed numerous cases of highly similar physical organization. Many of the sequences near rhg1 also showed significant similarity to Medicago and Arabidopsis. These results extend previous observations of large-scale duplication and selective gene loss in soybean, Medicago, and Arabidopsis, The results also suggest that conserved genome regions between Arabidopsis, Medicago, and soybean can stretch over long physical distances. By combining our results with other sequence data available in public databases, we were able to analyze a 340,000 base pair genome sequence region spanning rhg1. Through the use of informatic tools, all the tentative genes in this region have been annotated. Finally, we have adapted a new gene-silencing method for use in soybean, known as virus-induced gene silencing. Potentially, this technique will enable us to test numerous candidate genes quickly and efficiently for their effect on the plant's phenotype without the need for plant transformation.

Impacts
One of the most important diseases of soybean is the cyst nematode (SCN). In recent years, it became clear that one gene, rhg1, is especially important in resistance. My lab has worked to characterize this gene, pinpoint its location on the soybean map, and develop tools to isolate it by positional cloning. In the process, we have identified resistance genes elsewhere in the soybean genome, developed DNA markers suitable for marker-assisted breeding, and developed a broad range of genome tools for use in soybean and other legume crops.

Publications

• Foster-Hartnett, D., Mudge, J., Danesh, D., Yan, H., Larsen, D., Denny, R., Young, N.D. (2002) Comparative genomic analysis of sequences sampled from a small region on soybean molecular linkage group G. Genome 45:634-645.
• Penuela, S., Danesh, D., Young, N.D. (2002) Targeted isolation, sequence analysis, and physical mapping of nonTIR NBS-LRR genes in soybean. Theoretical and Applied Genetics 104:261-272.
• Zhu, H., Cannon, S., Young, N.D. Cook, D. R. (2002) Phylogeny and genomic organization of the TIR and non-TIR NBS-LRR resistance gene family in Medicago truncatula. Molecular Plant-Microbe Interaction 15:529-539.
• Cannon, S.B., Zhu, H., Baumgarten, A.M., Spangler, R., May, G., Cook, D.R., Young, N.D. (2002) Diversity, distribution, and ancient taxonomic relationships within the TIR and non-TIR NBS-LRR resistance gene subfamilies. Journal of Molecular Evolution.54:548-562.

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

Outputs
MARKER-ASSISTED SELECTION FOR SOYBEAN CYST NEMATODE (SCN) RESISTANCE. One of the most immediate applications of our genomic analysis of SCN resistance has been the development of several medium-throughput DNA markers suitable for use in marker-assisted breeding. As a result of this work, several Minnesota-adapted genotypes were tested as part of variety trials. These lines are part of our continuing effort to introduce new sources of SCN resistance into Minnesota soybeans. Potentially, the use of different donor genotypes can lead to greater durability of resistance over the long-run. During this period, intermediate and advanced lines were screened primarily on the basis of DNA markers, along with targeted greenhouse tests. We also continued to advance a special set of populations derived from a cross between Minnesota-adapted parents and PI 437654, the most SCN resistant genotype known. Four relatively large populations were tested through the use of marker-assisted selection in an effort to pinpoint exactly which lines retained resistance as well as promising agronomic characteristics. GENOMIC ANALYSIS OF RHG1. We completed our analysis of the soybean genomie in the region surrounding the rhg1 SCN resistance locus. This region is represented as nine contigs of nine or more bacterial artificial chromosomes (BACs) containing a total of1.2 Mb of unique soybean DNA. Approximately 150 sequences from BAC ends and subclones were determined and compared to Arabidopsis and M. truncatula. We also analyzed approximately 100 sequences derived from BAC contigs in duplicated regions of the soybean genome. One case of extensive soybean/M .truncatula/Arabidopsis microsynteny was examined in detail. In two other cases, BAC analysis indicated nearly identical sequence organization among duplicated regions of the soybean genome. Taken together, these results provide insight into the structure and organization of soybean and identify rules for assembling contigs from this complex genome.

Impacts
One of the most important diseases of soybean is the cyst nematode(SCN). Recently, it became clear that one gene, rhg1, is especially important in resistance. My lab has worked to characterize this gene, pinpoint its location on the soybean map, and develop tools to isolate it by positional cloning. In the process, we identified resistance genes elsewhere in the soybean genome, developed DNA markers suitable for marker-assisted breeding, and discovered many other interesting genes physically linked to rhg1.

Publications

• Chen, S. Y., Orf, J.H., Reese, C.D., Porter, P.M., Stienstra, W.C., Young, N.D., Walgenbach D., Schaus, P.J., Arlt, T.J., Breitenbach, F.R. 2001. Soybean cyst nematode population development & associated soybean yields of resistant & susceptible cultivars in MN. Pl Dis, 85:760-766.
• Marek, L.F., Mudge, J., Darnielle, L., Grant, D., Hanson, N., Paz, M., Huihuang, Y., Denny, R., Larson, K., Foster-Hartnett, D., Cooper, A., Danesh, D., Larsen, D., Schmidt, T., Staggs, R., Crow, J.A., Retzel, E., Young, N.D., Shoemaker, R.C. 2001. Soybean genomic survey: BAC-end sequences near RFLP and SSR markers. Genome 44: 472-581.

Progress 01/01/00 to 12/31/00

Outputs
MARKER-ASSISTED SELECTION FOR SOYBEAN CYST NEMATODE (SCN) RESISTANCE. Five Minnesota-adapted genotypes were tested as part of the final stages of regional variety trials. These lines were part of our contining effort to introduce new sources of SCN resistance into Minnesota soybeans, even as we pursue excellent agronomic and yield traits. Potentially, the use of different donor genotypes can lead to greater durability of resistance over the long-run. During this period, intermediate and advanced lines were screened primarily on the basis of DNA markers, along with targeted greenhouse tests. It was encouraging that greenhouse tests generally agreed with the results of DNA marker analysis. We also continued to advance a special set of populations derived from a cross between Minnesota-adapted parents and PI 437654, the most SCN resistant genotype known. Four relatively large populations were tested through the use of marker-assisted selection in an effort to pinpoint exactly which lines retained resistance as well as promising agronomic characteristics. GENOMIC ANALYSIS OF RHG1. We expanded our analysis of soybean linkage group G in the region surrounding the rhg1 SCN resistance locus. This region is represented as nine bacterial artificial chromosome (BAC) contigs containing a total of1.2 Mb of unique soybean DNA. Approximately 150 sequences from BAC ends and subclones were deterimined and compared to Arabidopsis thaliana and Medicago truncatula. We also analyzed approximately 100 sequences derived from BACs in duplicated regions of the soybean genome. One case of extensive soybean/ M truncatula/Arabidopsis microsynteny was observed. In two other cases, BAC analysis indicated nearly identical sequence organization among duplicated regions of the soybean genome. Taken together, our results provide insight into the structure and organization of soybean and identify rules for assembling contigs from this complex genome.

Impacts
One of the most important diseases of soybean is the cyst nematode(SCN). Recently, it became clear that one gene, rhg1, is especially important in resistance. My lab has worked to characterize this gene, pinpoint its location on the soybean map, and develop tools to isolate it by positional cloning. In the process, we identified resistance genes elsewhere in the soybean genome, as well as other interesting genes physically linked to rhg1.

Publications

• Young, N. D. (2000) The genetic architecture of resistance. Current Opinion in Plant Biology 3: 285-290.

Progress 01/01/99 to 12/31/99

Outputs
Marker-Assisted Selection for SCN Resistance. During this period, we made substantial progress in our first true marker-assisted selection (MAS) screening of soybean lines. Nearly 2,000 soybean lines were assayed with our best DNA marker, Satt309. Of these, several hundred lines were double-checked in the greenhouse and, except for one test that gave unusual results, SCN reactions were confirmed more than 80% of the time. The DNA marker assay took a few days, while the greenhouse assays took weeks. These observations parallel the results of a multi-year, field-based study in which MAS-selected lines were tested for impact on SCN eggs densities and yield. We initiated a focused MAS effort on the most promising source of SCN resistance, PI 437654. Lines from a cross between this genotype and a Minnesota adapted parent were screened with Satt309 plus, Satt187, which tags a second resistance gene. Ten F3 lines that carry both genes will be sent to Chile for increase. Traditional crossing and breeding continued with approximately 20 new crosses made this summer and more than 5,000 F2 individuals grown out. We released a new SCN resistant variety (MN0902) that is a late maturity group 0 suitable for planting in central/northern Minnesota. Positional Cloning of SCN Resistance. We pinpointed the major gene for SCN resistance, rhg1, to a genetic interval with more than 30 well-characterized DNA markers. It was feasible to begin the process of chromosome walking. We completed the construction of a new BAC library for soybean, as well as a preliminary screen for BAC clones located very near rhg1. Several clones nearby were uncovered. Because genetic transformation of soybean is difficult and time-consuming, we examined the A. rhizogenes/Ri root transformation as a system to quickly test candidate rhg1 sequences. The logic behind these experiments was that resistant cultivars grown in culture and transformed with a reporter Ri construct should continue to show the resistance phenotype, while susceptible cultivars should behave as susceptible. The results confirmed this expectation. The results provided insight into the localization of Ri-insert expression, as well as the activity of different promoters. We sought to discover putative resistance gene sequences from throughout the soybean genome. We hoped to find the rhg1 gene (without chromosome walking), as well as candidates for many other resistance genes of interest in soybean. While we have not yet discovered the rhg1 gene itself, we have found hundreds of "resistance gene analogs" . Comparative Genomic Analysis. We have been investigating parallels in chromosome organization between the rhg1 region and homologous regions within soybean and the model legume system, Medicago truncatula. The goal of this work is to uncover similarities and differences in the way the genome is organized in related regions. In the process, we have found long stretches of DNA sequences that show remarkable levels of similarity. This type of information enables us to make predictions about how the soybean genome might have evolved at the molecular level.

Impacts
One of the most important diseases of soybean is the cyst nematode(SCN). Recently, it became clear that one gene, rhg1, is especially important in resistance. My lab has worked to characterize this gene, pinpoint its location on the soybean map, and develop tools to isolate it by positional cloning. In the process, we identified resistance genes elsewhere in the soybean genome, as well as other interesting genes physically linked to rhg1.

Publications

• Cregan, P., Mudge, J., Fickus, E. W., Danesh, D., Denny, R.L., Young, N. D. (1999) Two simple sequence repeats to select for soybean cyst nematode resistance conditioned by the rhg1 locus. Theoretical and Applied Genetics.99(5):811-818.
• Cregan, P., Mudge, J., Fickus, E. W., Marek, L. F., Danesh, D., Denny, R. Shoemaker, R. C., Matthews, B., Young, N. D. (1999) Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes. Theoretical and Applied Genetics. 98: 919-928.
• Meyers, B. C., Dickerman, A. W., Michelmore, R. W., Pecherer, R. M., Sivaramakrishnan, S., Sobral, B. W., Young, N. D. (1999) Plant disease resistance genes encode members of an ancient and diverse protein family within the nucleotide-binding superfamily. The Plant Journal. 20: 317-332.
• Narayanan, R.A., Atz, R., Denny, R.L., Young, N.D., Somers, D.A. (1999) Expression of soybean cyst nematode resistance in transgenic hairy roots of soybean. Crop Science. In press
• Young, N. D. (1999) A cautiously optimistic vision for marker-assisted breeding. Molecular Breeding. 5: 505-510.

Progress 01/01/98 to 12/31/98

Outputs
In previous work, we have shown that resistance to soybean cyst nematode (Heterodera glycines; SCN) is controlled primarily by a major gene, known as rhg1. In the past year, we completed the development of breeder-friendly DNA markers to select for this gene and tested the efficacy of marker-assisted selection. The markers, which are based on "microsatellites," can be assayed on simple agarose gels without the need for radioactivity. In a pilot study, a single technician was able to test 1000 soybean lines in three weeks, a substantial increase in speed compared to traditional greenhouse SCN-screening methods. In detailed comparisons between markers and greenhouse phenotype, DNA markers were more than 95% accurate in identifying susceptible lines in the University of Minnesota breeding program.

Impacts
(N/A)

Publications

• Cregan, P., Mudge, J., Fickus, E. W., Marek, L. F., Danesh, D., Denny, R.Shoemaker, R. C., Matthews, B., Young, N. D. (1998) Targeted isolation of simple sequence repeat markers through the use of bacterial artificial chromosomes. Theoretical and Applied Genetics. In press.

Progress 01/01/97 to 12/31/97

Outputs
In previous work, we have shown that resistance to cyst nematode (Heterodera glycines; SCN) in soybean is controlled primarily by a major gene located at the top of molecular linkage group G. This locus is commonly referred to as rhg1. In the past year, we have focused on the development of breeder-friendly DNA markers to select for this gene. Using a DNA isolation method based on a solid-matrix method that can process 96 samples at once, we have optimized several DNA markers tightly linked to rhg1. These markers, which are based on microsatellites (also known as simple sequence repeats), can be assayed on simple agarose gels without the need for radioactivity. Using this combination of DNA-isolation and microsatellite markers, a single scientist can now determine the SCN phenotype for more than 500 individuals in just one week. This represents a substantial increase in throughput compared to previous SCN-screening methods. We are now integrating this approach to SCN resistance selection into our soybean breeding program at the University of Minnesota.

Impacts
(N/A)

Publications

• Lange, D.A., Penuela, S., Denny, R.L., Mudge, J., Orf, J.H., Young, N.D. 1998. A plant DNA isolation protocol suitable for polymerase chain reaction marker-assisted selection. Crop Science: in press.
• Mudge, J., Cregan, P.B., Kenworthy, J.P., Kenworthy, W.J., Orf, J.H. Young, N.D. 1997. Two microsatellite markers that flank the major soybean cyst nematode resistance locus. Crop Science 37: 1611-1615.

Progress 01/01/96 to 12/30/96

Outputs
Through the use of DNA markers, we have characterized the genes for cyst nematode (SCN) resistance in soybean. In previous work, we determined that the most important resistance gene is located on linkage group 'G'. In the past year, we extended this work to identify minor genes that modulate the resistance. In two soybean lines often used in breeding for SCN resistance (PI 209332 and PI 90763), we determined that a second gene is located on linkage group 'J' of soybean. In another resistant soybean line ('Peking') we uncovered a second resistance gene on linkage group 'G' and another on linkage grou 'N'. Significantly, all of the SCN resistance genes uncovered to date are located at or near the locations of other soybean resistance genes (including several fungal, bacterial, and viral pathogens). To apply our knowledge about SCN resistance in practical plant breeding, we also developed an improved method for isolating soybean DNA. This method involved immobilizing the DNA on a solid matrix, followed by processing in a 96-well microplate. A single person can isolate and purify DNA, as well as initiate PCR-based analyses, for 96 individuals in one day. The DNA samples are suitable for several polymerase chain reaction DNA amplification procedures. Becasue the DNA is immobilized on a solid matrix and processed in 96-well plates, this protocol could easily be modified for robotic manipulation.

Impacts
(N/A)

Publications

• Concibido, V.C., Lange, D.A., Denny, R.L., Orf, J.H. and Young, N.D. (1997). Genome mapping of soybean cyst nematode resistance loci in 'Peking', PI 90763, and PI 88788 using DNA markers. Crop Science: In press.
• Denny, R., Lange, D., Penuela, S., Mudge, J., Orf, J. and Young, N. (1996) Marker-assisted selection for soybean cyst nematode resistance. Soybean Genetics Newsletter 23:179-182.
• Mudge, J., Concibido, V., Denny, R., Young, N. and Orf, J. (1996) Genetic mapping of yield depression locus near a major gene for soybean cyst nematode resistance. Soybean Genetics Newsletter 23:175-178.
• Orf, J. and Young, N. (1996) Registration of 'Freeborn' soybean. Crop Sci. In press. Young, N. (1996) QTL mapping and complex disease resistance in plants. Annual Review of Phytopathology 34:479-501.

Progress 01/01/95 to 12/30/95

Outputs
The primary target of our work on multigenic disease resistance in plants has been cyst nematode of soybean (Heterodera glycines; SCN). This is the most important disease problem of soybean, but resistance is complex & progress in this area has been slow. Our approach, which is to use DNA genetic markers to dissect the individual components of resistance, has demonstrated that there is a major resistance gene located near the top of linkage group 'G' of soybean. This resistance gene is shared by at least 5 different sources of SCN resistance & controls up to 50% of total variation in resistance. Along with this major gene, two other SCN resistance genes are of special note. A second resistance gene on linkage group 'G' is also highly effective, but is found primarily in the resistant soybean genotype, 'Peking'. A third resistance gene on linkage group 'J' is shared by soybeans PI209332 and PI90763. This gene is highly effective against SCN. It is also interesting in that it is located in a genomic region of soybean that contains four additional genes involved in host-parasite interactions. Separately, we have shown that an important yield-depression gene is located close to the major SCN resistance gene at the top of linkage group 'G'. Linkage between SCN resistance & low yield is a major problem for farmers, so we are using DNA markers to identify promising lines in which this undesirable linkage has been broken. The results of this effort may be ready for field trials within a year-two.

Impacts
(N/A)

Publications

• BOUTIN, S., YOUNG, N., LORENZEN, L., AND SHOEMAKER, R. 1995. Color DNA marker pedigrees and graphical genotypes generated by Supergene software. Crop Science: 35:1703-1707.
• BOUTIN, S., YOUNG, N., OLSON, T., YU, Z.H., SHOEMAKER, R. AND VALLEJOS, C. 1995. Genome conservation among three legume genera detected with DNA markers. Genome 38:928-937.
• CONCIBIDO, V., DENNY, R., LANGE, D., DANESH, D., ORF, J., AND YOUNG, N. 1995. DNA marker analysis and linkage mapping of soybean cyst nematode resistance. Proceedings of SCN Res. Conf. (Ames, IA, March 1995) p. 17-20.
• CONCIBIDO, V., DENNY, R., LANGE, D., DANESH, D., ORF, J., AND YOUNG, N. 1995. The Soybean cyst nematode resistance gene on linkage group G is common among sources of resistance. Soybean Genetics Newsletter. 22:269-272.
• YOUNG, N., CONCIBIDO, V., DANESH, D., DENNY, R., BOUTIN, S. AND LANGE, D. 1995. Genome mapping in legumes:insights into molecular evolution, gene mapping, and positinal cloning. Plt. Genome III (San Diego, CA, 1/1995), p. 13.

Progress 01/01/94 to 12/30/94

Outputs
My research program uses genome mapping techniques to study disease resistance in soybean and examine relationships in genome organization between soybean and its relatives. We have identified DNA markers that are very close to resistance loci for soybean cyst nematode (SCN), one of the most severe limits to soybean productivity in the U.S. Currently my lab is using marker-assisted breeding to develop soybean varieties that carry better forms of SCN resistance. Our research demonstrates that at least five unlinked genes play a role in SCN resistance and one locus on linkage group G is especially significant in its impact on resistance. With this genetic mapping data as a foundation, we are developing improved cultivars more quickly than would be possible with conventional breeding alone. Genome mapping also enables us to dissect the roles of individual genes involved in this complex disease resistance trait. In related work, we are examining the chromosomal organization of soybean in relation to other legume species. Since the genome of soybean is relatively large, physical analysis of the soybean genome is difficult. However, two related legume crops (Vigna and Phaseolus) have smaller genomes more amenable to physical mapping. We have found that there are several conserved chromosomal blocks providing a basis for studying molecular evolution in legumes and speeding efforts at (SCN resistance) gene cloning based on genetic map position.

Impacts
(N/A)

Publications

• BOUTIN, S., OLSON, T., YU, Z., YOUNG, N., SHOEMAKER, R., AND VALLEIJOS, E. 1994. Substantial genome conservation among legume genera detected with DNA markers. Plant Genome II (San Diego, CA, January 1994).
• CONCIBIDO, V., DENNY, R., BOUTIN, S., HAUTEA, R., ORF, J., AND YOUNG, N. 1994. DNA marker analysis of loci underlying resistance to soybean cyst nematode (Heterodera glycinea Ichinohe). Crop Science 34:240-246.
• CONCIBIDO, V., DENNY, R., DANESH, D., ORF, J., AND YOUNG, N. 1994. High resolution mapping & race specificity of a partial resistance gene for SCN.Proc. 5th Biennial Conf. on Molecular & Cellular Bio. of Soybean. Athens, GA. p. 3.
• YOUNG, N. 1994. Constructing a genetic linkage map with DNA markers, In: DNA Based Markers in Plants. R. Phillips and I. Vasil, eds. Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 39-57.
• YOUNG, N. 1994. Plant gene mapping, In: Encyclopedia of Agricultural Science Vol. 3. C. Arntzen, Ed. Academic Press, San Diego, pp. 275-282.
• YOUNG, N. AND PHILLIPS, R. 1994. Cloning plant genes known only by phenotype (meeting report). Plant Cell 6:1193-1195.

Progress 01/01/93 to 12/30/93

Outputs
To characterize disease resistance in plants and pathogenecity in disease-causing organisms, my lab uses a set of gene mapping tools known as restriction fragment length polymorphism (RFLP) markers. With RFLPs, genome organization can be examined, important genes can be mapped and molecular genetic relationships among organisms explored. Our research has focused on soybean, mungbean and two disease pathogens, soybean cyst nematode (SCN) and the bean rust fungus. Mungbean is an especially useful organism for studying legumes because of its small genome size and rapid lifecycle. In research on SCN resistance, we identified 3 major genes. While each individual gene controls only a fraction of the phenotype, together, the genes control more than 60% of total variation. Further studies have demonstrated that one of the resistance genes shows a differential response to SCN isolates from different states, despite the fact that the isolates are the same pathogen race. By contrast, another resistance gene acts independent pathogen isolate. A similar project in mungbean to uncover partial resistance genes against powdery mildew is also underway. Using RFLPs and a genetic marker known as Random Amplified Polymorphic DNAs (RAPDs), relationships among isolates of bean rust and SCN have been examined. In bean rust, the bean cultivar where a sample was isolated from appears to be an important determinant in relatedness, as is the ability to form sexual structures.

Impacts
(N/A)

Publications

• CONCIBIDO, V., DENNY, R., BOUTIN, S., HAUTEA, R., ORF, J., YOUNG, N. (1994) DNA marker analysis of loci underlying resistance to soybean cyst nematode (Heterodera glycinea Ichinohe). Crop Science, in-press.
• FATOKUN, C., DANESH, D., YOUNG, N., STEWART, E. (1993) Molecular taxonomic relationships in the genus Vigna based on RFLP analysis. Theoretical and Applied Gen 86: 97-104.
• MENANCIO-HAUTEA, D., FATOKUN, C., KUMAR, L., DANESH, D., YOUNG, N. (1993) Comparative genome analysis of mungbean (Vigna radiata (L.) Wilczek) and cowpea (V. unguiculata (L.) Walpers) using RFLP mapping data. Theoretical and Applied Gen.
• YOUNG, N., KUMAR, L., MENANCIO-HAUTEA, D., DANESH, D., TALEKAR, N., SHANMUGASUNDARUM, S., KIM, D. (1992) RFLP mapping of a major bruchid resistance gene in mungbean (Vigna radiata, L. Wilczek). Theoretical and Applied Genetics 84:839-844..
• YOUNG, N., DANESH, D., MENANCIO-HAUTEA, D., KUMAR, L. (1993) Mapping oligogenic resistance to powdery mildew in mungbean with RFLPs. Theoretical and Applied Genetics 87:243-249.
• FATOKUN, C., MENANCIO-HAUTEA, D., DANESH, D., YOUNG, N. (1992) Evidence for orthologous seed weight genes in cowpea and mungbean based on RFLPs. Genetics 132:841-846.

Progress 01/01/92 to 12/30/92

Outputs
To understand complex disease resistance traits in plants, special genetic mapping techniques are required. My research program uses genetic markers known as Restriction Fragment Length Polymorphisms (RFLPs) and Random Amplified Polymorphic DNAs (RAPDs) to characterize complex disease resistances in soybean and its relatives, as well as pathogenicity in their pathogens. Research in soybean focuses on resistance to the cyst nematode, Heterodera glycines. Previous work has demonstrated that several independent resistance genes are involved. Using DNA markers, my associates and I have successfully mapped the chromosomal locations of two major genes (and two minor genes) for cyst nematode resistance. Together, these resistance genes explain more than 50% of the total variation in resistance. Now, we are putting the results to practical use to develop an improved soybean variety that carries the essential resistance genes with a minimum of undesirable traits from the original (and wild) resistant soybean parent. At the same time, complex resistances in a relative of soybean, Vigna (mungbeans and cowpeas), are also being examined. Vigna is extremely helpful in understanding genome organization in legumes because of its very small genome size. So far, resistance genes for a gemini virus, the powdery mildew fungus, and the pod weevil have all been mapped in Vigna.

Impacts
(N/A)

Publications

• BOUTIN, S., ANSARI, H., CONCIBIDO, V., DENNY, R., ORF, J. and YOUNG, N. 1992. RFLP analysis of cyst nematode resistance in soybeans. Soybeans Genetics Newsletter 19:123-127.
• BOUTIN, S., LORENZEN, L., SHOEMAKER, R. and YOUNG, N. 1992. Computer generated graphical genotypes and DNA marker pedigrees. Proceedings of the Fourth Biennial Conference on Molecular and Cellular Biology of the Soybean. Ames, Iowa.
• CONCIBIDO, V., BOUTIN, S., DENNY, R., ANSARI, H., ORF, J. and YOUNG, N. 1992. RFLP mapping of cyst nematode resistance genes in soybean (Glycine max). Proceedings of the Fourth Biennial Conference on Molecular and Cellular Biology.
• FATOKUN, C., MENANCIO-HAUTEA, D., DANESH, D. and YOUNG, N. 1992. Evidence for orthologous seed weight genes in cowpea and mungbean based on RFLPs. Genetics, 132:841-846.

Progress 01/01/91 to 12/30/91

Outputs
As a basis for studying the genetics of disease resistance in legumes, my laboratory has developed DNA marker maps for the genus, Vigna. This system was chosen because it has the smallest genome (set of chromosomes) of all legumes, which makes it especially attractive for molecular analysis and gene cloning. The Vigna map has also been compared with that of soybean to determine the degree of commonality. In the process, we have mapped the chromosomal locations of several disease and pest resistance genes. In particular, we have mapped the location of a single dominant gene conferring resistance to a serious insect pest known as bruchids. This gene is an ideal target for molecular cloning based on chromosome walking technology. Moreover, we have also mapped a set of three unlinked genes controlling a complex foorm of resistance to the fungus, Erysiphe polygoni (powdery mildew). Current experiments are focusing on mapping genes for resistance to several important legume viruses and the fungal pathogen, Cercospora leaf spot. In parallel experiments, DNA markers are being used to map the locations of genes for resistance to the cyst nematode of soybean (Heterodera glycines). Cyst nematode resistance is controlled by several genes and we have already determined the putative locations for some of these genes.

Impacts
(N/A)

Publications

Progress 01/01/90 to 12/30/90

Outputs
In order to clone genes from legumes and legume pathogens that control host-parasite interactions, my associates and I are constructing genetic linkage maps composed of restriction fragment length polymorphisms (RFLPs). We have chosen the legume genus, Vigna, which has a small and simple genome that is excellent for molecular genetic analysis, and the legume pathogen, Uromyces appendiculatus (bean rust) as model systems for study. The linkage map of Vigna now consists of 50 RFLP markers, of which 34 have already been placed on seven linkage groups covering 300 centimorgans. Two loci that control resistance to the powdery mildew, Erysiphe polygoni, have been tagged with nearby RFLPs. We have also identified another powdery mildew resistance gene in Vigna that shows an unusual tissue-specific mode of resistance. Ultrastructural analysis indicates that the basis of this resistance may be significantly lower numbers of stomata on the upper surface of the leaf. RFLP mapping of the genes controlling resistance and stomate number is now underway. In separate work, we have begun RFLP mapping in Uromyces. First, we developed a rapid DNA extraction protocol to isolate large amounts of DNA from the parents and progeny of a Uromyces population segregating for several avirulence genes. We are now preparing nucleic acid blot filters that will be used to assess DNA sequence variation between the parents and construct an RFLP linkage map for this organism.

Impacts
(N/A)

Publications

• YOUNG, N., MESSEGUER, R., GOLEMBOSKI, D. and TANKSLEY, T. (1991) DNA near disease resistance genes in tomato. In: Resistance to Viral Disease of Vegetables: Genetics and Breeding. M. Kyle, ed. Timber Press. in-press.
• YOUNG, N., FATOKUN, C., DANESH, D. and MENANCIO-HAUTEA, D. (1991) RFLP mapping in cowpea. Proceedings of the Workshop, Biotechnology: Enhancing Research on Tropical Crops in Africa. in-press.
• YOUNG, N., MENANCIO-HAUTEA, D., FATOKUN, C. and DANESH, D. (1991) RFLP technology, crop improvement, and international agriculture. Proceedings of the Workshop, Biotechnology; Enhancing Research on Tropical Crops in Africa. In Press.
• YOUNG, N., DANESH, D., GIESER, P. (1990) RFLP mapping, gene tagging, and DNA fingerprinting In Vigna. Proceedings of the International Research Meeting of the Bean/Cowpea Collaborative Research Support Program. (Abstract).
• YOUNG, N. (1990) Potential applications of Map-based cloning to plant pathology. Physiological and Molecular Plant Pathology 37: 81-94.