Bradeen Lab – University of Minnesota Lab Goal: Development of “allelic mining” techniques and strategies for R genes, enabling multi-genotype isolation of R gene alleles. 1. Optimization of long range PCR (LR-PCR) for recovery of R gene orthologs 2. Testing / Demonstration of LR-PCR method for RB locus 3. Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity: ● Geographic distribution ● Morphological variation ● Within vs. between population relationships ● Genome-wide diversity ● Mating system 4. Development of PCR approaches for R gene homolog recovery Complete On-going Lab Objectives:

Objective 1: Optimization of long range PCR (LR-PCR) for recovery of R gene orthologs (summary of previously reported research) Towards improved LR-PCR, we evaluated: ● six genomic DNA extraction methods ● multiple LR-PCR primers for RB ● five LR-PCR (Taq) systems ● two cloning systems Criteria for evaluation: ● amplification of target sequence ● minimal amplification of non-target ● sequence fidelity (low error rates) Status: complete 3 presentations at national / international meetings accepted (in press): Molecular Breeding

Objective 2: Testing / Demonstration of LR-PCR method for RB locus (update of previously reported research) Hypotheses from previous research: ● #1: RB (functional allele) is frequent occurring ● #2: RB is Type II (slow evolving, little paralog interchange) We used improved LR-PCR methods for RB recovery in 44 S. bulbocastanum genotypes; successful amplicon in 17 genotypes (~40% success). Conclusion highlights (details on following slides): ● Intron and LRR sequence confirms recovery of orthologs (true alleles) ● RB (functional) and rb (non-functional) alleles predominate (consistent with hypothesis #1) ● Identified rc-RB, an unique recombinant allele: traumatic paralog rearrangement; ~3kb insert of unknown origin; event occurred in planta (refutes hypothesis #2)

Objective 2: Testing / Demonstration of LR-PCR method for RB locus (update of previously reported research) Intron and LRR sequence confirms recovery of orthologs (true alleles) RGA1RBRGA 3 RGA4 RBtr INTRON LRR Clade RB resistance allele Clade rb susceptibilit y allele LRR

Objective 2: Testing / Demonstration of LR-PCR method for RB locus (update of previously reported research) RB (functional) and rb (non-functional) alleles predominate Clade RB resistance allele Clade rb susceptibility allele LRR

Objective 2: Testing / Demonstration of LR-PCR method for RB locus (update of previously reported research) Identified rc-RB, an unique recombinant allele: traumatic paralog rearrangement; ~3kb insert of unknown origin; event occurred in planta RGA 1RGA 3RBRGA 4 Rc-RB

Objective 2: Testing / Demonstration of LR-PCR method for RB locus (update of previously reported research) Status:final analyses on-going presented at 2 national / international conferences manuscript to be submitted (in ~6 weeks): ● Plant Journal or ● Molecular Plant Microbe Interactions Future Directions: PCR characterization of Rc-RB distribution characterization of 3kb unique insert

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (update of previously reported research) ● Geographic distribution ● Morphological variation ● Within vs. between population relationships Nested within Objective 2 RB LR-PCR on 44 S. bulbocastanum genotypes: 3 morphologically- defined subspecies 12 populations multiple genotypes/pop Entire geographic distribution

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (update of previously reported research) Clade RB resistance allele Clade rb susceptibility allele LRR Low levels of RB allelic diversity; no obvious correlation with: ● Geographic distribution ● Morphological variation ● Within vs. between population relationships

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Newly reported research) ● Genome-wide diversity “Can we predict genome-wide diversity?” “Does greater genome-wide diversity = greater R gene diversity?” AFLP analysis (1-3 primer pairs) of 151 S. bulbocastanum genotypes ● entire geographic distribution ● 3 morphologically-defined subspecies ● 44 populations (2-5 genotypes/population) ● includes genotypes used in RB allelic mining study

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Newly reported research) Global Conclusion: S. bulbocastanum, as a species, lacks clear genetic structure; suggests extensive gene flow

ssp. bulbocastanum ssp. dolichophyllum ssp. partitum Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Newly reported research)

Geographic locale Subspecies classification Within vs. between population WithinBetween AMOVA (partitions observed molecular variation): 87.65%12.35% 87.25%12.75% 84.66%15.34% Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Newly reported research)

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Newly reported research) S. bulbocastanum lacks clear genetic structure: geographic origin, morphological variation, and within vs. between population relationships do not predict genetic diversity Although we uncovered little RB allelic diversity, RB and rb were found from genotypes throughout our dendrogram: total genome diversity does not predict RB allelic diversity “Does greater genome-wide diversity = greater R gene diversity?” “Can we predict genome-wide diversity?” ● Genome-wide diversity

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Newly reported research) Status:complete presented at 3 national / international conferences manuscript submitted (Dec 2005): Molecular Breeding Future Directions: effects of mating system (see following slides) ● Genome-wide diversity

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Proposed research) Results of RB allelic mining and AFLP diversity analyses are consistent with extensive gene flow throughout S. bulbocastanum. S. bulbocastanum is an obligate allogamous (outcrossing) species Consistent with NSF Potato Genome Project goals for R gene allelic mining throughout the genus Solanum, we will pursue genome-wide (AFLP) and RB homolog diversity (see Objective 4) analyses in S. polyadenium, an obligate autogamous (selfing) species. ● Mating system

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Proposed research) S. polyadenium is resistant to late blight (left) and Verticillium (right). ● diploid, autogamous ● closely related to S. bulbocastanum ● potential for potato improvement

Objective 3: Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity (Proposed research) Sampling approach: Entire Potato Genebank collection Entire geographic distribution Multiple genotypes/population Data to be generated: AFLP RB homologs (Objective 4) Experimental Plan ● Mating system

Objective 4: Development of PCR approaches for R gene homolog recovery (New, ongoing, and proposed research) Impetus: Advisory Committee, 2005 “…sample more easily amplified fragments of the genes…” Our response: We are targeting the informative RB LRR and intron regions. These regions are amplified from genomic DNA using primers generic to all RB paralogs, allowing isolation of RB homologs (note different focus than Objective 2), from multiple genotypes with emphasis on S. bulbocastanum, S. polyadenium, and more distantly related Solanaceous species. We are testing approaches to “fingerprint” amplified fragments for targeted sequencing, reducing overall costs: Ecotilling, DGGE

Objective 4: Development of PCR approaches for R gene homolog recovery (New, ongoing, and proposed research) Intron (~860bp): ● divergent between paralogs ● used to define RB “alleles” LRR (~1,685bp): ● divergent between paralogs ● divergent between RB alleles ● subtle changes = big effect (?) INTRON LRR

Objective 4: Development of PCR approaches for R gene homolog recovery (New, ongoing, and proposed research) Fingerprinting approaches (to be combined with targeted sequencing): 1. Ecotilling D11186A13

Objective 4: Development of PCR approaches for R gene homolog recovery (New, ongoing, and proposed research) Fingerprinting approaches (to be combined with targeted sequencing): 1. Ecotilling 2. DGGE: Denaturing Gradient Gel Electrophoresis PCR products separated based on sequence (GC content) Commonly used for microbial fingerprinting of environmental samples Samples will be fingerprinted and representatives sequenced.

Progress to Date: 1. Optimization of long range PCR (LR-PCR) for recovery of R gene orthologs 2. Testing / Demonstration of LR-PCR method for RB locus 3. Germplasm selection strategy for maximal diversity: characterize potential predictors of R gene allelic diversity: ● Geographic distribution ● Morphological variation ● Within vs. between population relationships ● Genome-wide diversity ● Mating system 4. Development of PCR approaches for R gene homolog recovery Complete On-going Lab Objectives: Bradeen Lab – University of Minnesota

Progress to Date: Bradeen Lab – University of Minnesota 7 presentations at national / international meetings 1 manuscript in press (Molecular Breeding) 1 manuscript submitted (Molecular Breeding) 1 manuscript to be submitted soon (Plant Journal, MPMI)

Plans for the next 12 months: 1. completion of RB ortholog study 2. exploration of distribution of rc-RB and of unique 3kb region 3. AFLP analysis of S. polyadenium: effects of mating system 4. RB homologs: optimization of fingerprinting strategy Bradeen Lab – University of Minnesota