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

Cosegregation of CAPS with Disease Phenotypes in Barley
Ehren Whigham1, John Upah1, Greg Fuerst2,4, Matt Moscou3, Karin Werner2,4, Liu Xi3, Jasmine Chen3, 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
Discussion
Abstract
Research Statement
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 F2 (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 F2 population and was screened
for co-segregation of one of the identified CAPS and the disease
phenotypes.
To identify polymorphic markers in parental barley strains and screen
the F2 population for co-segregation of a marker with the disease
phenotype.
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.
Methods
Three Mla6 mutants were independently
crossed with Morex and an F2 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 F2
population to generate amplicons for digest.
Digest data and phenotype data were
compared to identify co-segregation of a
maker with the disease phenotypes.
Results
Phenotype Data:
x2
Mutant
m11542
wt
37
sp
9
n
6
sp & n
0
6.88
p-value
0.076
M9467
36
8
8
1
3.33
0.343
Expected
9
3
3
1
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 F2 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 cosegregate with the disease phenotypes.
Wild type
References
Susceptible
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.
Cell Death
Genotype Data:
Mla6 Morex
Example of progression from
parental PCR digestion with
HF01G22T to F2 population PCR with
the same primer set, to F2 population
digest with Bgl II.
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.
Acknowledgement
F2 Population PCR
F2 Population Restriction Digest
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.