Transcript Dennis Vaughn1,John Jackson1, Matt Moscou24,Karin Werner24

Cosegregation of Phenotypes with Genotypes in OWB
Ehren Whigham1, Lance Maffin1, Greg Fuerst2,4, Weihui Xu3, Karin Werner2,4, Charles Warwick3, and Roger Wise3,4
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
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.
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.
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.
Hood Phenotype (R)
Awn Phenotype (D)
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.
Kap Amplicon
References
D R D R R R D D R R R R D D R D D R D D
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 Iclass homeobox gene. PNAS 104(4): 1424-1429.
Methods
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.
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.
Acknowledgement
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
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.