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

Identification of Pathogen Defense Genes in Cereal Plants
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Ehren Whigham1, John Upah1, Greg Fuerst2,4, Matt Moscou3, Karin Werner2,4, Liu Xi3, Amy Schwartz3, 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
Research Hypothesis
This project was designed to identify the knockout genes responsible
for causing two mutant strains of barley to be susceptible to powdery
mildew. Based on bioinformatic data, primers were designed to target
genes thought to be involved in defense. PCR was performed to
locate the deleted gene in both mutants. Simultaneously, an F2 (wild
type x mutant) population was grown and phenotypes were recorded
for several days after inoculation with the Blumeria graminis f. sp.
hordei (Bgh) isolate cc148 to identify individuals displaying the mutant
and wild type phenotypes. No deletions were identified, the F2
generation was screened for co-segregation. This screen supports the
deletion as responsible for causing susceptibility.
To locate the deleted gene in mutants of C.I. 16151, and to determine
if the deleted gene co-segregates with the mutant phenotype.
Results
No deletions were found in either mutant (m9467 or m9468) after PCR
screening 48 candidate genes under three annealing temperatures:
52, 55, and 58ºC.
m9467, 52oC annealing temperature
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.
o
m9468, 52 C annealing temperature
Analysis of the PCR results for m9467 and m9468
using the designed primers yielded no deletions in
either mutant. Possible explanations include no
deletion present at the target location, multiple copies
of the gene in the genome, and ineffective primer
design.
Phenotype data compiled for both mutants indicates
which plants express the mutant phenotype and
which express the wild type phenotype. This data will
be useful when screening for co-segregation after the
gene deletions for both mutants are identified.
Future work includes the design of new primer sets
and redesign of existing primer sets. When the
deletion is found, screening of the F2 population for
co-segregation will be possible.
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.
Methods
DNA was extracted from two fast-neutron
derived barley mutants (m9467 and m9468)
and the wild-type (C.I. 16151) using 2X CTAB.
Sample concentrations were determined
using a spectrophotometer and diluted for use
as template in subsequent PCR reactions. To
find possible gene deletions within the
mutants, PCR was conducted at multiple
annealing temperatures, and possible
deletions were repeated using a gradient
thermocycler. To determine if any deletions
co-segregated with the inoculated mutant
phenotypes, F2 generation plants were
subjected to powdery mildew and phenotyped
for necrosis and tip wilt. DNA was extracted
for all individuals of these populations using
the methods described above.
Discussion
Nine individuals from the m9467 x C.I. 16151 F2 population exhibited
the mutant phenotype of necrosis (cell death) and tip wilt. Similarly,
six individuals from the m9468 x C.I. 16151 F2 population exhibited
the mutant phenotype.
C.I. 16151
m9467
m9468
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.
Transcript Based Cloning of Rom1
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Dennis Vaughn1,John Jackson1, Matt Moscou2,4,Karin Werner2,4,Yan Meng3, Pingsha Hu3, Rico Caldo3 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
This project was designed to locate the knockout gene
responsible for causing two mutant strains of barley to
be susceptible to powdery mildew. After bioinformatics
analysis, primers were designed to target genes thought
to be involved in defense. PCR was performed to
locate the deleted gene in both mutants. Simultaneously,
an F2 (wild type x mutant) population was grown and
phenotypes were recorded for several days after
inoculation with fungus to identify mutant and wild type
individuals. After a deletion was identified, the F2
generation was screened for co-segregation. This
screen supports the deletion as responsible for causing
Background
susceptibility.
The most common plant defense against pathogen
attack involves the entire plant being resistant to a
specific pathogen. In other cases the plant resistance is
gene specific. The resistance to powdery mildew in
barley has been found to be gene specific in some
cases. For example, the Rar1 gene was identified to
cause resistance in Sultan V (Freialdenhoven et al 2005).
However, a mutation of the rar1 gene in Sultan V caused
susceptibility to pathogens in M100. It is believed that a
further mutation between M100 and rom1 caused
resistance again. Using Transcript Based Cloning we
can determine if a mutation occured Sultan V to M100.
Methods
After harvesting several varieties of Barley,
DNA was extracted and then prepared for
amplification using the CTAB Fresh Frozen Prep.
Next the selected genes were amplified using PCR
with Designed Rom1 and M100 The amplified
genes were inserted in a vector and cloned using
ligation and transformation. After cloning the
cultures were plated on ampicillin agar. Success
colonies were then picked and a mini Prep was
used to prepare for sequencing.
Design
PCR
primers
PCR
Extract
DNA from
tissue
QIAquick PCR Purification / Gel Extraction
Transformation
Plating
Picking Colonies
PCR Validation
Mla12
M100
rom1
(Rar1 Rom1)
(rar1 Rom1)
(rar1 rom1)
We identified differentially expressed
genes between M100 and rom1.
Fourteen genes were physically mapped
to chromosome 2(2H). We utilized the
ELP algorithm MarkerMaker to place
6,425 genes on the genetic map. Two
genes were identified that mapped
proximal to the centromere from marker
MWG503 and were differentially
expressed between m100 and rom1. We
are currently sequencing PCR products
and will continue our investigation.
Centromere
distance
(cM)
E3546-13
Rom1, E4044-5
map position
MWG503
281 (190)
2.1
0.2
Ant2
MWG892
0.7
0.015
Rar1, MWG87
MWG876
2.2
0.8
cMWG694
MWG2123
Telomere
References
Freialdenhoven, Andreas. (2005) Barley Rom1 Reveals
a Potential Link Between Race-Specific and Nonhost
Resistance to Powdery Mildew Fungi.
Overnight Cultures
Mini-Prep
Sequence
After amplifying Rom1 candidate gene using the
designed primers eight samples were selected for
cloning as illustrated in Figure 1. Eventually,
three were picked from plating to be sequence.
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To determine if the gene identified as the deletion is
responsible for the necrosis phenotype associated with
susceptibility to white powdery mildew.
NaN3
Ligation
Results
Research Question/Hypothesis
NaN3
Discussion
Cho, Seungho. (2006) Transcriptome Analysis and
Physical Mapping of Barley Genes in Wheat-Barley
Chromosome Addition Lines
Acknowledgement
I would like to thank the Plant Genomics
Outreach Program at Iowa State University
especially to Adah Leshem-Ackerman and
Jonathan Wendel for their support. In addition, I
would like to thank the Biotechnology Outreach
Education Center and the Office of
Biotechnology for their generous assistance
with equipment, teaching and support.
Mostly, I would like thank the members of the
Wise Lab including my mentor Roger Wise, Rico
Caldo, Yan Meng, Pingsha Hu, Karin Werner, and
especially Matt Moscou for their support,
guidance, and patience.