1. Cosegregation of CAPS with Disease Phenotypes in Barley Abstract This project was designed to identify cleaved amplified polymorphic sequence (CAPS) markers in two strains of barley. Primers for candidate CAPS were generated based on prior research. PCR was performed to identify which markers were polymorphic. Simultaneously, an F 2 (Morex x mutant Mla 6) population was grown and phenotypes were recorded for several days after inoculation with the Blumeria graminis f. sp. hordei (Bgh) isolate cc5874 to identify individuals displaying the disease and wild type phenotypes. DNA was extracted from each individual in the F 2 population and was screened for co-segregation of one of the identified CAPS and the disease phenotypes. Methods Background Plant diseases are one of the greatest problems to crop production worldwide. Genomic research such as gene deletion studies provide the information necessary to control these diseases (Zhang, 2006). Previously, fast neutron mutagenesis was conducted on wild type (C.I. 16151) seeds to randomly knock out chunks of genomic DNA. The progeny of these seeds were then planted and inoculated with the powdery mildew isolate 5874 (Blumeria graminis f. sp. hordei). Plants displaying cell death symptoms or sporulating colonies were selected. Seeds from these plants were then planted and inoculated with the same fungal isolate. RNA samples were collected at six time points after inoculation and hybridized to a Barley1 GeneChip (Close et. al. 2003). Bioinformatic analysis was conducted to determine the genes, based on expression patterns, most likely to have been knocked out in m9467 and m9468. Primers were designed for those 48 genes and PCR (polymerase chain reaction) was used to identify the deletions. Results Research Statement Discussion References Close, TJ, S Wanamaker, R Caldo, SM Turner, DA Ashlock, JA Dickerson, RA Wing, GJ Muehlbauer, A Kleinhofs and RP Wise. 2004. A new resource for cereal genomics: 22K barley GeneChip comes of age. Plant Phys. 134:960-968. Zhang, L, T Fetch, J Nirmala, D Schmierer, R Brueggeman, B Steffenson, and A Kleinhofs. 2006. Rpr1, a gene required for Rpg1-dependent resistance to stem rust in barley. Theor. Appl. Genet. 113:847-855. Ehren Whigham 1, John Upah 1, Greg Fuerst 2,4, Matt Moscou 3, Karin Werner 2,4, Liu Xi 3, Jasmine Chen 3, and Roger Wise 3,4 1 - Secondary Biology Teacher Intern, Iowa State University Plant Pathology, Iowa State University, Ames, IA 50011 2 - Technician-USDA, Department of Plant Pathology, Iowa State University, Ames, IA 50011 3 - Department of Plant Pathology and Center for Plant Responses of Environmental Stresses, Iowa State University, Ames, IA 50011 4 - Corn Insects and Crop Genetics Research, USDA-ARS, Iowa State University, Ames, IA 50011 Acknowledgement We would like to thank the Plant Genomics Outreach Program at Iowa State University especially Adah Leshem-Ackerman and Jay Staker for their support. In addition, we would like to thank the Biotechnology Outreach Education Center and the Office of Biotechnology for their generous assistance with equipment, teaching and support. Mostly, we would like thank the members of the Wise Lab including our PI Roger Wise, Matt Moscou, Karin Werner, and especially Greg Fuerst for all of their support, guidance, and patience. Phenotypes for m11542 and m9467 segregated in a 9:3:3:1 ratio, wt: sp: n: sp&n. The other mutant, m11524 showed an interesting and unexpected dwarf phenotype and analysis was put on hold until an explanation could be found. A total of 16 CAPS were identified from a pool of 96 candidates. 2 more from another project were added for a total of 18 CAPS. The F 2 populations of m11542 and m9467 were screened with 9 of the 18 identified CAPS. Digest and phenotype data will be analyzed to determine if any of the screened markers co- segregate with the disease phenotypes. Three Mla6 mutants were independently crossed with Morex and an F 2 population was grown. The plants were observed and phenotypes recorded for several days after innoculation. Disease phenotypes include resistant (wt), susceptible (sp), and cell death (n). DNA was extracted from all three populations and each parent using 2X CTAB. PCR was done on parental DNA to generate amplicons that were digested with restriction enzymes to identify CAPS markers. Based on the identified CAPS, PCR was done on the F 2 population to generate amplicons for digest. Digest data and phenotype data were compared to identify co-segregation of a maker with the disease phenotypes. Phenotype Data: Mutantwt sp n sp & n x 2 p-value m1154237 9 6 0 6.88 0.076 M946736 8 8 1 3.33 0.343 Expected 9 3 3 1 To identify polymorphic markers in parental barley strains and screen the F 2 population for co-segregation of a marker with the disease phenotype. Wild type Susceptible Cell Death Genotype Data: Example of progression from parental PCR digestion with HF01G22T to F 2 population PCR with the same primer set, to F 2 population digest with Bgl II. Mla6 Morex F 2 Population PCR F 2 Population Restriction Digest