iPath – Extending iTAG Barley into Plant-Microbe Interactions for Grade 9-12 ClassroomsSarah Michaelson1, Julie Gonzalez2, Greg Fuerst3,4, and Roger Wise3,4

1 – Science Teacher, Westover School, Middlebury, CT 067622 – Biotechnology Program Chair, Des Moines Area Community College, Ankeny, IA 50236

3 – Plant Pathology and Microbiology, Iowa State University, Ames, IA 500114 – Corn Insects and Crop Genetics Research Unit, USDA-ARS, Iowa State University, Ames, IA 50011

Introduction

The iTAG Barley Module was developed in 2009 by teachers participating in the Research Experience for Teacherprogram in collaboration with the Roger Wise laboratory at Iowa State University. The module has since been usedsuccessfully in high school and college classrooms to provide students the opportunity to use hands-on laboratorytechniques and develop an understanding of the relationships between genotype and phenotype. This poster willdescribe the work conducted this summer to create an extension to the iTAG module. The iPath extension exposesstudents to concepts of plant pathology and the connection between plant genetics and resistance or susceptibilityto plant pathogens. Cereal crops including wheat, barley, and rye are affected by a variety of pathogens, which canreduce yields by 30% to 40%. One of the most economically important cereal pathogens is powdery mildew,Blumeria graminis. The iPath module will allow students to extract DNA and isolate the Mla6 gene, which confersresistance to B. graminis. Students can then compare their gene expression data with their observations of plantphenotypes.

Discussion

The challenge in designing the iTAG and iPath modules was making complex concepts accessible to high schoolstudents. To address this challenge, the iTAG and iPath modules use a subset of the Oregon Wolfe Barley (OWB)population consisting of double haploid lines which have either all dominant or all recessive genes for several easilyidentifiable phenotypes, making the analysis of genotypes and phenotypes easily understandable for students.Similarly, primers had to be designed and selected based on which primer sets would result in gel patterns that wouldbe most clearly understood by high school students. The primers designed in this project for the resistance gene,Mla6, will be effective for use in the high school classroom. Primers designed in the divergent region of the geneshow a clear presence and absence pattern on a gel, while primers designed in the conserved region show identicalstrong bands in each lane of the gel. Instructors can use these results to inform student understandings of genestructure and alleles.

The concepts presented in the iPath module will provide a real-world relevance to many topics covered in atraditional high school biology course. DNA structure, genotypes and phenotypes, host-pathogen relationships, andco-evolution are all topics that teachers could address by using this module. The methodology of the module alsoallows students to practice the science and engineering practices emphasized in the Next Generation ScienceStandards.

Acknowledgements

We would like to thank the Research Experience for Teachers program, and coordinator Adah Leshem, along with theDepartment of Plant Pathology and Microbiology of Iowa State University. In addition, we would like to thank themembers of our sponsoring lab – Dr. Roger Wise and Greg Fuerst - for their support, guidance, and patience.

Funded by NSF-PGRP 0922746 & 1339348

Results

Purpose

Design an activity for high school students to isolate and analyze pathogen-resistant genes in barley.Design primers in conserved and divergent regions of the resistant gene.

C

Methods

The Oregon Wolfe Barley Informative and Spectacular Subset (OWB ISS) were inoculated with powdery mildew. Afterone week of incubation the lines were analyzed for the presence of an active powdery mildew infection and scored asresistant or susceptible. Next, NCBI was used to find sequences for seven alleles of the Mla gene, which confersresistance to powdery mildew. The seven gene sequences were aligned using the Multiple Sequence Alignment Toolfrom the European Bioinformatics Institute. Based on the alignment, primers were designed in both the conservedand divergent regions of the gene, and Integrated DNA Technology’s OligoAnalyzer software was used to optimizeprimer sets. To amplify the genes from the OWB ISS, DNA was extracted from each line and PCR was performed withthe conserved and divergent primer sets. Gel electrophoresis was used to visualize and compare gene expressionbetween the twenty lines of barley.

Figure 3.A. Resistant to powdery mildew (S. Michaelson)B. Susceptible to powdery mildew (S. Michaelson)C. Close up of powdery mildew colonies on barley

leaves (P. Spanu)

Figure 1.Oregon Wolfe Barley Informative and Spectacular Subset photographed seven days after inoculation. Phenotypes for resistance and susceptibility are indicated parenthetically.

A

B

Figure 4.A. Gel results of Mla6 PCR with divergent

region primersB. Gel results of Mla6 PCR with conserved

region primers

A B A

B

A

B

Figure 2.A. A sample conserved region of alignmentB. A sample divergent region of alignment