Vernalization Gene Architecture as a
Predictor of Growth Habit in Barley
• Douglas Heckart
• Primary Advisor: Dr Patrick Hayes
• Secondary Advisor: Dr Thomas Chastain
• Project Advisor (field): Ann Corey
• Project Advisor (laboratory) Dr Peter Szucs
Overview
• Vernalization background
• Phenotype determination methods
• Genotype determination methods
• Relating genotype to phenotype
Purpose
• Validate candidate genes for winter and facultative
growth habit in barley.
• Important to OSU barley
breeding program for
development of fall sown
malting barley with cold
tolerance.
• Determine if growth habit can be predicted from
vernalization genotype
Winter Hardiness
A function of:
• Low temperature tolerance
• Photoperiod
• Vernalization
Vernalization Background
• Vernalization: Cold temperature required for
the vegetative to reproductive transition in an
agronomically acceptable time frame.
Required by winter barleys such as Strider
Barley growth habit types as related
to vernalization requirement
•Winter: Vernalization required and cold tolerant
•Facultative: No vernalization required and cold tolerant
•Spring: No vernalization
required and cold sensitive
Subject of study since 1970
Vernalization Genetics
•
Two Locus epistatic
model
• VrnH1 on Chromosome
5H
• Candidate: HvBM5A
• VrnH2 on Chromosome
4H
• Candidate: ZCCT-H
present or deleted
Chromosome
4H
ZCCT-H encodes a
repressor, a DNA
binding protein
Chromosome 5 H
• HvBM5A is a meristem identity
gene that can have a binding site
for the repressor encoded by
ZCCT-H.
Winter genotype
4H Chromosome
Repressor
5H Chromosome
Repressor Binding
Site
ZCCT-H
HvBM5A
TRANSCRIPTION BLOCKED THEREFORE FLOWERING IS REPRESSED
Winter genotype
5H Chromosome
4H Chromosome
Repressor
Repressor Binding
Site
ZCCT-H
HvBM5A
= TRANSCRIPTION OCCURS AND FLOWERING INITIATED
6 C° ~ 6 wks
4H Chromosome
Facultative
genotype
5H Chromosome
Repressor Binding
Site
ZCCT-H
deleted
HvBM5A
TRANSCRIPTION OCCURS
= No vernalization requirement, but low
temperature tolerant
Spring genotype
5H Chromosome
4H Chromosome
Gene present
without binding
site
ZCCT-H
HvBM5A
TRANSCRIPTION OCCURS = FLOWERING INITIATED
Spring genotype
5H Chromosome
4H Chromosome
Gene present
without binding
site
ZCCT-H
deleted
HvBM5A
TRANSCRIPTION OCCURS = FLOWERING INITIATED
Von Zitzewitz et al. (2005)
Relates Growth
Habit to Vrn
Genotype
• Winter types require
vernalization because:
•They contain the ZCCT-H (repressor) gene and
the HvBM5A “winter” allele with a repressor
binding site
Von Zitzewitz et al. (cont.)
• Spring types require no vernalization
because:
•They contain HvBM5A allele with a
deletion: no repressor binding site
•ZCCT-H gene may or
may not be present
Von Zitzewitz et al. (cont.)
• Facultative types do not require vernalization
because:
• Deletion of ZCCT-H and
• Presence of the HvBM5A winter allele with a
repressor binding site
Methods
and
Materials
Phenotype determination
• Plant growth staging using Feekes growth
stage scale
• Each assessment was performed on 54
genotypes – fall-sown and spring-sown
Feekes Growth Stages
• Assessed every 14 days
•
On a plot basis
•
Terminated at Feekes
10.5
Feekes Growth Stages
Feekes Growth Stages
Growing Degree Day Vernalization
Coefficient (GDD-VC)
•
Growing Degree Days (GDD) from planting to Feekes
10.5 for fall and spring sown experiments
• GDD was calculated using a 10°C base
•
GDD-VC = spring GDD – fall GDD. GDD = 1,000 for
lines that did not flower in the spring-sown nursery
Genotyping procedures
•
54 lines in the ORELT
genotyped at two locations:
USDA/ARS lab at Pullman,
WA and OSU Barley
project lab at Corvallis, OR
•
HvBM5A dominant PCRbased marker
• ZCCT-H a dominant PCRbased marker
Results
Variety or
Fall sown
Spring sown
selection
GDD
GDD
GDD-VC
Kold
285
1000
715
88 AB 536
122
424
302
Orca
113
319
206
Results
35
Facultative
30
Winter
Number of Lines
25
20
15
10
5
0
139
236
302
409
Vrn requirement GDD-VC
715
812
Results
 100% agreement for HvBM5A markers
 All entries are therefore winter or facultative
Rechecking ZCCT-H
= Discrepant with Pullman Data
Results
ZCCT-H discrepancies between labs
 Dominant marker
 Considered the presence of an amplicon at
one location to be evidence of gene
presence
Results
• 7 lines failed to amplify at USDA/ARS lab
that amplified at the OSU lab
• 2 lines failed to amplify at the OSU lab
that amplified at the USDA/ARS lab
Results
Variety
Fall sown
Spring sown
GDD
GDD
GDD-VC
Genotype
Maja
188
424
236
F
88 AB 536
122
424
302
F
Kold
285
1000
715
W
Strider
188
1000
812
W
3 lines violate the model
ORELT
Variety or
Fall
Spring
GDD-
growth habit
no.
Selection
GDD
GDD
VC
classification
18
J2-6-19
188
424
236
W
23
StabBC 50-9-1
188
424
236
W
24
StabBC 50-7-1
285
424
139
W
Conclusions
•The model was validated: 51 out of 54 lines fit
•Accurately predict growth habit from marker
data ~ 94% of the time
•The Vernalization phenotype is a suitable target
for marker assisted selection (MAS)
Explanation for not having 100% fit:
•Other “maturity” genes possibly play a role in
determination of spring/winter phenotypes
•DNA contamination
•Ambiguity in rating the phenotype
•Residual heterogeneity
Residual Heterogeneity
Line #23
Heading occurred, but
sparse
Heterogeneity apparent
Recommendations for future research
• Improve genotyping
•Augment the Vrn-H2
genotyping with a codominant
assay.
•HvSnf2 is tightly linked to
ZCCT-H and present in all
growth habit types
Recommendations for future research
• Improve phenotyping
• Record growth stages daily
• Count Final Leaf Number on
ambiguous lines
Acknowledgments
I would like to thank:




Dr. Hayes for his extreme patience and help along the way
Dr. Chastain for his guidance and encouragement
Ann Corey for all the help with the field portion of this experiment
Peter Szucs for the many hours of help in the lab. He took my
project seriously and was an excellent teacher
 The Hayes barley lab at Oregon State University for providing
answers to my questions
 The Bioresource Research program for providing an opportunity to
undertake such a project as an undergraduate
 The Crop and Soil Science Department at Oregon State
University. I would hate to think what school would have been like
without my CSS family. Thank you very much!