Cosegregation of Phenotypes with Genotypes in OWB

1. Cosegregation of Phenotypes with Genotypes in OWB Abstract The purpose of this research is multifaceted. The first objective was to identify polymorphisms in the sequence of two identified genes associated with obvious phenotypes in an Oregon Wolfe Barley (OWB) population. The second objective was to transfer laboratory research methods, techniques and experiences to a high school classroom setting. This required modification of several protocols containing toxic/hazardous chemicals . It also involved designing activities that scaffolded to the modified research protocols to create understanding. The third objective was to find a mechanism to distinguish dominant from recessive PCR amplicons of the vrs1 gene. Methods Background Oregon Wolfe Barley are double haploid cultivars bred to have either all dominant or all recessive genes for specific phenotypic traits. This makes study of Mendelian inheritance through a population easy to study given that the phenotypes chosen are easily distinguishable and measurable even to high school students. One gene of interest used in the teaching module is is vrs1. This gene controls whether the seed spike contains two or six viable rows of seed. The second gene of interest is the Kap gene, which determines the presence/absence and length of the awn. Using these two genes of interest a teaching module has been constructed to address cosegregation of a specific genotype with a specific phenotype. This concept is not only important in plant pathology, but is foundational to all areas of genetics including human trait inheritance and medicine. Ehren Whigham1, Lance Maffin1, Greg Fuerst2,4, Weihui Xu3, Karin Werner2,4, Charles Warwick3, and Roger Wise3,41 - Secondary Biology Teacher Intern, Iowa State University Plant Pathology, Iowa State University, Ames, IA 500112 - Technician-USDA, Department of Plant Pathology, Iowa State University, Ames, IA 500113 - Department of Plant Pathology and Center for Plant Responses of Environmental Stresses, Iowa State University, Ames, IA 500114 - Corn Insects and Crop Genetics Research, USDA-ARS, Iowa State University, Ames, IA 50011 Discussion Research Statement To screen a population of Oregon Wolfe Barley for cosegregation of phenotypes with genotypes and to transfer that research into a secondary education classroom teaching module. Cosegregation was verified for the Kap gene and the hooded vs. awned phenotype. PCR generated 1500bp and 2000bp bands representing the recessive (hooded) and dominant (awn) alleles respectively. Cosegregation was verified for the vrs1 gene following PCR and subsequent digestion using restriction enzyme NciI. The vrs1 gene controls the phenotype of two row or six row seed spikes. Digestion generated a 734bp band for the recessive (six row) allele, whereas the dominant (two row) allele displayed a 468bp band. Hood Phenotype (R) Awn Phenotype (D) Kap Amplicon 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. McCoy, SB. 2000. Understanding epistasis in linkage analysis: the kap and lks2 loci in the Oregon Wolfe Barley population. BS Thesis, Oregon State University. Komatsuda, T. et. al. 2007 Six-rowed barley originated from a mutation in a homeodomain-leucine zipper I-class homeobox gene. PNAS 104(4): 1424-1429. D R D R R R D D R R R R D D R D D R D D Acknowledgement We would like to thank the Plant Genomics Outreach Program at Iowa State University especially Adah Leshem-Ackerman for her 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. We would also like to thank Dr. Pat Hayes for providing Kap and vrs1 primer sequence and information regarding the OWB population. Mostly, we would like thank the members of the Wise Lab including our PI Roger Wise, Weihui Xu, Karin Werner, Julie Meyer, Charles Warwick and especially Greg Fuerst for all of their support, guidance and patience. A subpopulation of Oregon Wolfe Barley, including the parents, was grown for DNA extraction. Leaf tissue was collected at two weeks and DNA was extracted using a nontoxic protocol that can be spread over several 45 minute class periods. PCR was done to amplify both the Kap and vrs1 genes for the entire population. Restriction digestion of the vrs1 amplicon using NciI was required to differentiate between dominant and recessive genotypes. Phenotypic data was compared with genotypic data and screened for cosegregation.. Simple procedures including a strawberry DNA extraction and a pipette tip box electrophoresis lab will be used to scaffold student understanding of extraction and gel electrophoresis. Six Row Seed Spike Phenotype (R) Two Row Seed Spike Phenotype (D) Predicted Fragment Lengths as a Result of Nci1 Vrs1 Amplicon after Digestion with NciI D R R R R R R D D D R D R D D R R D R D