AN ABSTRACT OF THE THESIS OF
Jarislav von Zitzewitz for the degree of Master of Science in Crop Science presented
on September 23, 2004.
Title: Structural and Functional Characterization of Candidate Vernalization Genes in
Barley
Abstract approved:
Redacted for privacy
-
Patrick
Vernalization
yes
the requirement of a period of low temperature to induce the
transition from a vegetative to a reproductive state
is an evolutionarily and
economically important trait in the Triticeae. The genetic basis of vernalization in
barley (Hordeum vulgare subsp. vulgare), a model crop for the Triticeae, was first
defined in terms of a three-locus epistatic model. Candidate genes for two of the
vernalization loci (Vrn] and Vrn2) were recently cloned in diploid and polyploid
Triticurn species. The sequence and expression of HvBM5A, a candidate for Vrn-H1,
were characterized in a panel of ten barley germplasm accessions, including one
accession of the wild progenitor (Hordeum vulgare subsp. spontaneum). Differences
in vernalization requirement are most likely due to allelic variation at the HvBM5A
promoter andlor intragenic sites rather than in the coding region. An analysis of
promoter and intron 1 polymorphisms showed that differences in the sequence of the
latter are most consistent with growth habit classifications. HvBM5A expression
patterns correlate with growth habit and meristem development. HvBM5A maps to the
predicted position of Vrn-H1 on chromosome 5H. All available evidence suggests that
HvBM5A is Vrn-H1. The presence/absence of the tightly linked ZCCT-H gene family
members on chromosome 4H is perfectly correlated with growth habit in the
germplasm array: all spring forms show a complete deletion of the ZCCT-H gene
family. All available evidence suggests that a ZCCT-H gene family member is VrnH2. All QTL for vernalization requirement reported for barley in the literature can be
explained by a two-locus model involving the Vrn-H1 and Vrn-H2 candidates. The
data from this study provide a rigorous definition for "facultative growth habit":
facultative genotypes have the Vrn-H2 deletion and signature "winter" habit alleles at
Vrn-H1. These data lay the foundation for determining if the coincidence of Vrn-H1
with QTL for multiple winter hardiness-related phenotypes is due to linkage and/or
pleiotropy.
©Copyright by Jarislav von Zitzewitz
September 23, 2004
All Rights Reserved
Structural and Functional Characterization of Candidate Vernalization Genes in Barley
by
Jarislav von Zitzewitz
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Presented September 23, 2004
Commencement June 2005
Master of Science thesis of Jarislav von Zitzewitz presented on September 23, 2004
APPROVED:
Redacted for privacy
Major Professor, representii'ig Crop Sence
Redacted for privacy
Head of the Department of Crop and Soil Science
Redacted for privacy
Dean of th9'ra4Iate School
I understand that my thesis will become part of the permanent collection of Oregon
State University libraries. My signature bellow authorizes release of my thesis to any
reader upon request.
Redacted for privacy
Jarislav von Zitzewitz, Author
/
ACKNOWLEDGEMENTS
My gratitude is expressed to my major professor Dr. Patrick M. Hayes, for his
guidance, encouragement, and understanding during critical times throughout the
course of my study. I would also like to thank him for his hospitality and for sharing
times in his back yard with family, BBQ, and beer. And thanks for a few good
sessions of Oregon surf
I further express my appreciation to the other members of my committee, who
have so graciously given of their time and expertise: Dr. Tony H.H. Chen, Dr. Isabel
Vales, and Dr. Oscar Riera-Lizarazu. Special thanks to Dr. Jeffrey Skinner for his
excellent counsel and guidance on the design, conduct, and analysis of this study. I
would also like to thank him for the good times we have had mountain biking.
My sincere appreciation is extended to the members of the OSU barley project:
First of all I would like to thank my friend Ariel Castro, for encouraging me all the
way, and as a roommate, teacher of history and politics of my own country; a thanks
to Tanya Filichkin, who always helped in all aspects of the laboratory and always was
a good listener; Dr. Jennifer Kling, who was always there for any sort of statistical
problem; Dr. Lol Cooper, as a weekend laboratory companion; Dr. Luis MarquezCedillo, who has been here since the beginning and shared so much with me; and my
graduate colleague Kelly Richardson, who has always helped when I asked for it. I
would like to specially thank my Hungarian friend, Dr. Peter Szucs, who helped
tremendously with this research, for his camaraderie and all the laughter and beers we
shared. Another special thanks to Ann Corey, who also has been here since the
11
beginning, for her friendship and for being so kind in hosting me at her home with her
family during the last moments of my thesis writing.
Many thanks to Alexa Michel, who helped in early morning sessions of (cold
room) apical meristem collections. Special thanks to Dr. Hiro Nonogaki and his Ph.D.
student Po-Pu Liu for providing RNA extraction protocols and helping in designing
expression studies. A note of thanks to Zoran Jeknic who showed me the "ins and
outs" of the digital camera, and Caprice Rosato and Mark Dasenko for assisting me
with gene sequencing.
This research was supported by the National Science Foundation and the North
American Barley Genome Project. Without this support, this work would have never
been possible.
I would like to extend my thanks to my Portland friends, Sara and Ryan
Retzlaff, who became very good friends to me and have helped so much in my
personal life since I began my education at Oregon State University in 1997.
Finally, and most important, a thanks to my parents, brother and sisters, who
have supported me throughout my studies; my in-laws Jorge and Pilar, and sisters in
law for having me at their home through difficult transitional times; and infinite thanks
to my beautiful wife Cecilia and daughter Manuela, who without their patience,
support, kind words of encouragement, and lots of love, I would have never been able
to complete this work.
111
CONTRIBUTION OF AUTHORS
Dr. Patrick M. Hayes, initiated, advised, and supervised all aspects of this
project. He contributed in formulating hypothesis and in preparation of this
manuscript. Dr. Jeffrey S. Skinner made substantial contributions to the preparation of
this manuscript, and he provided essential assistance in the design and execution of
experiments. Dr. Peter Szucs aided in cloning and sequencing alleles, genotyping, and
analysis of data. Dr. Tony H. H. Chen helped in advising, manuscript revision, and
experimental design. Dr. Jorge Dubcovsky and Liuling Yang provided valuable prepublication data and information, and participated in manuscript preparation.
lv
TABLE OF CONTENTS
Page
General Introduction
.1
Structural and Functional Characterization of Candidate
Vernalization Genes in Barley ........................................................................................ 8
ABSTRACT ........................................................................................................ 9
INTRODUCTION............................................................................................ 10
RESULTS......................................................................................................... 14
DISCUSSION ................................................................................................... 37
MATERIALS AND METHODS ...................................................................... 45
LITERATURE CITED ..................................................................................... 53
General Conclusions ..................................................................................................... 57
Bibliography .................................................................................................................. 62
Appendix ....................................................................................................................... 67
Figure Al. Alignment of the HvBM5A coding sequence in
ten barley varieties differing in growth habit .................................................... 71
Figure A2. Alignment of the HvBM5A promoter sequence in ten
barley varieties differing in growth habit .......................................................... 77
LIST OF FIGURES
Page
Figure
1.
Linkage map positions of HvBM5A, HvBM5B and inferred
position of ZCCT-H in the Dicktoo x Morex population .................................. 16
2. Exonlintron structure of Morex HvBM5A and HvBM5B and
primerpositions ................................................................................................. 17
3. Alignment of predicted peptides of fIvBM5A and other MADS-box
AP1/SQUAMOSA family members ................................................................. 21
4. Northern blots of HvBM5A in Strider (winter), Dicktoo (facultative)
and Morex (spring) (A); contrast enhanced images of Strider and
Dicktoo (B); and RT-PCR of HvBM5A and HvBM5B in Strider,
Dicktoo and Morex (C) ..................................................................................... 24
5. Apical meristems of Strider, Dicktoo, and Morex sampled at specific
time points and treatments shown in expression studies (Figure 4) .................. 26
6. HvBM5A promoter (-2 kb) haplotypes in winter/facultative and
spring genotypes vs. Tremois and OSU 11 with predicted motifs(A)
and predicted TATA-box and the putative CArG motif (B) ............................. 28
7. HvBM5A Intron 1 length polymorphism in winter, facultative, and
spring barley genotypes (A) and sequence alignments of a conserved
portion of intron 1 for HvBM5A, TaVRT1 and TmApl(B) ................................ 32
8. PCR amplification of SnJ2 and ZCCT-H showing deletion in
facuttative and spring genotypes ....................................................................... 34
vi
LIST OF TABLES
Table
Page
1.
Conserved HvBM5A promoter elements found in ten barley genotypes
that could be involved in vernalization requirement or abiotic stress
responses (see Figure 6A for relative positions) ............................................... 30
2. HvBM5A intron 1 elements found in a 436 bp region conserved
between Hordeum and Triticum accessions, involved in flowering
or abiotic stress responses ................................................................................. 33
3.
Summary of Vrn-H1 and Vrn-H2 allele types compared to vernalization
requirement QTL reported in the literature ....................................................... 37
Structural and Functional Characterization of Candidate Vernalization genes in
Barley
General Introduction
Vernalization can be defined as the induction of flowering by exposure of a
plant to extended periods of low temperature. The range of effective temperatures at
which flowering is promoted by cold is usually between 1-7°C, and in some cases
vernalizing temperatures can be as low as -6°C (Michaels and Amasino, 2000).
Vernalization is characteristic of many temperate zone plants, including "winter
growth habit" forms of the Triticeae. Candidate genes for the Vrn-A1 and Vrn-A2 loci
were recently cloned in diploid wheat (Triticum monococcum) (Yan et at., 2003 and
2004) and in the A, B, and D genomes in hexaploid wheat (Triticum aestivum)
(Danyluk et al., 2003; Trevaskis et al., 2003; Murai et al., 2003). This has considerably
advanced our understanding of this trait in economically important crop species. The
genetics of vernalization are well-characterized in Arabidopsis thaliana (see review by
Henderson et al., 2003; Henderson and Dean, 2004), where the central genes in the
vernalization pathway are now known to be different than the central genes in the
Triticeae (Yan et at., 2003 and 2004). Recently, it has been shown that genes in the
Arabidopsis thaliana vernalization pathway are involved in epigenetic silencing by
histone modifications (Sung and Amasino, 2004; Bastow et al., 2004). In the
Triticeae, such detailed studies are still in progress.
2
In the Triticeae, vernalization is an important trait due to its role in winter
hardiness, which can be defined as the capacity of a genotype to survive the winter. In
this simple definition, the complex genetic and genetic x environmental interactions
that transpire between planting and harvesting a winter cereal crop (Hayes et al.,
1997), need to be accounted for. Winter hardiness is composed of three principal
traits, which include low temperature tolerance, vernalization requirement, and
photoperiod sensitivity. The three phenotypes are usually associated, e.g. "winter
varieties" that are low temperature-tolerant usually require vernalization and are
photoperiod sensitive. The requirement of vernalization is the trait that is usually
associated the most with winter hardy genotypes.
However, in an extensive characterization of a range of barley (Hordeum
vulgare subsp. vulgare) gerrnplasm, Karsai et al., (2001) found that some of the most
low temperature-tolerant genotypes were photoperiod sensitive but did not require
vernalization. Such varieties are termed "facultative" because agronomically speaking,
they could be fall or spring-sown. The variety "Dicktoo", which is a model for winter
hardiness research in the barley community (Pan et al., 1994; van Zee et al., 1995;
Hayes et al., 1997; Choi et al., 2000; Mahfoozi et al., 2000; Karsai et al. 2001; Fowler
et al., 2001) shows facultative growth habit: it is cold tolerant, it is sensitive to short
photoperiod, and does not have a vernalization requirement. There is evidence in
wheat that maximum low temperature tolerance is associated with both vernalization
and photoperiod sensitivity (Mahfoozi et al., 2001). Therefore, it is important to
understand the structure and function of Triticeae vernalization genes. Determining
the genes and pathways involved in the vernalization pathway is of fundamental
scientific importance and has the potential to allow plant breeders to tailor varieties to
specific environments.
The Triticeae are a very useful tool to study the genetics of vernalization
because they form a homogeneous genetic system, in which results from one species
are frequently applicable to other members of the cereal tribe (Dubcovsky et al.,
1998). Barley (Hordeum vulgare subsp. vulgare) belongs to the Triticeae, and is an
economically important crop model for studying the genetics and physiology of
vernalization. Barley is a simple genetic system due to its self-pollinating and diploid
nature, and it displays genetic variation for the components of winter hardiness. In
addition, an ever-expanding set of tools exists for genetic and molecular analysis
(reviewed in Hayes et al., 2003), including multiple mapping populations, arrayed
BAC clones (Yu et al., 2000), a large EST database, and a microarray chip (Close et
al., 2004). These tools could be very useful to rapidly develop gene-specific markers
to improve elite lines during conventional and marker assisted breeding (Francki and
Appels, 2002).
The foundations for the genetics of vernalization requirement in barley were
established by Takahashi and Yasuda (1971), who proposed a three-locus epistatic
model in which genotypes that require vernalization (winter types) have the allelic
architecture
Sh sh2sh2sh3sh3.
All other allelic configurations lead to facultative and
spring growth habits. Based on wheat: barley orthology, the Sh loci are now described
using standard Triticeae nomenclature, with an "H" indicating the Hordeum genome,
4
as follows: Sh2 = Vrn-H1 (chromosome 5H); Sh = Vrn-H2 (chromosome 4H); and
Sh3= Vrn-H3 (chromosome 111). Allelic variants at the Vrn-H3 locus are reported
only in barleys from extreme high or low latitudes (Takahashi and Yasuda, 1971).
Yan et al. (2003, 2004) proposed a model explaining the Vrn2/Vrnl epistatic
interaction in the Triticeae, based on the positional cloning and expression studies of
Vrn-A1 and Vrn-A2 candidates in T. monococcum. Per this model, ZCCT1 (the
candidate gene for Vrn2) encodes a dominant repressor of flowering that binds to a cis
element regulatory region of Api (the candidate for Vrni) a MADS-box gene.
Vernalization down-regulates the expression of Vrn2 allowing expression of Vrni. A
lack of vernalization requirement occurs in genotypes with a deletion for Vrn2,
regardless of the allele at Vrni as well as in genotypes that do have Vrn2 but that lack
a target binding site for the repressor in Vrnl. A deletion was reported in the promoter
region of Vrn-A1 that correlated with spring vs. winter growth habit. The candidate
barley ortholog of Vrn-A1 was first described as barley MADS-box 5 (HvBM5) by
Schmitz et al. (2000), but the work was not related to vernalization and HvBM5 was
not equated with Vrn-H1. Additional evidence supporting the orthology of barley
HvBM5 with T monococcum Api (Vrn-Ai) is provided in the expression studies
reported by Danyluk et al. (2003), and Trevaskis et al. (2003).
Differences in the genetics of vernalization in Arabidopsis and the Triticeae
should be mentioned, because similar gene names used for both species do not imply
to have a common function. The Triticeae ApI candidate for Vrnl is similar to the
Arabidopsis meristem identity gene APE TA LA 1 (APi), but Arabidopsis APi is not
directly involved in vernalization (Yan et al. 2003; Trevaskis et al. 2003; Murai et al.,
2003). In Arabidopsis, the vernalization-requirement phenotype is conferred by a
combination of functional alleles at the FRIGIDA (FRI) and FLOWERING LOCUS C
(FLC) loci (Burn et al., 1993; Koornneef et al., 1994). ZCCT1 , the candidate for Vrn2,
is similar to FLC (as repressor), but belongs to a different family of transcription
factors and contains a putative zinc finger in the first exon and a CCT domain in the
second exon, in common with the Arabidopsis CONSTANS (CO) and CO-like proteins
(Yan et al. 2004). The CCT domain localizes CO in the nucleus (Robson et al. 2001),
the central gene in the mechanism by which day length controls flowering (Valverde
et al. 2004). CO and FLC proteins regulate SOC 1, a flowering time gene,
antagonistically via separate promoter motifs (Hepworth et al., 2002), suggesting that
transcription factors involved in vernalization and photoperiod target upstream genes
that are in common to both pathways. In Arabidopsis, Vrnl and Vrn2 are involved in
the repression maintenance of FLC, clearly distinguishing their role from the T.
monococcum genes with the same name. Obviously, the genetics of vernalization in
the model plant Arabidopsis is complex and certainly additional genes will be
identified in the Triticeae that are fundamental players in the vernalization pathway.
An interesting recent discovery is the control of FLC chromatin by histone
methylation and acetylation during vernalization (Sung and Amasino, 2004; Bastow et
al., 2004). These reports give a new insight on the complexity of vernalization and
provide leads to investigate in the Triticeae.
6
Due to the complexity in vernalization and its role in winter hardiness in the
Triticeae, quantitative trait locus (QTL) analysis tools have been used for trait
dissection, which was simplified in barley, a diploid. For example, Laurie et al. (1995)
reported vernalization QTL on chromosomes 411 and 5H in the Igri x Triumph (winter
x spring) mapping population and the QTL positions correspond to the predicted
locations of the Vrn-H2 and Vrn-H1 loci. Francia et al. (2003), reported a
vernalization QTL on 5H at the predicted position of Vrn-H1 in the Nure x Tremois
(winter x spring) mapping population. QTL for multiple winter hardiness-related
traits, including crown fructan content, photoperiod sensitivity and low temperature
tolerance (Hayes et al., 1993) were mapped in the Dicktoo x Morex (facultative x
spring) population on 5H that corresponds with the predicted position of Vrn-H1
(Karsai et al. 1997). The availability of multiple barley QTL mapping populations
segregating for components of winter hardiness provides a unique opportunity to test
the Vrnl/Vrn2 model of Yan et al. (2004) and to further explore the relationships of
vernalization, photoperiod sensitivity, and low temperature tolerance.
A longer-term objective in our breeding program is to completely characterize
the components of winter hardiness in barley. In this study we focused on
vernalization and asked the question: "does the Vrnl/Vrn2 epistatic model proposed
by Yan et al. (2004) for T. monococcum apply to barley?" Our specific goals were to
determine if (i) allelic variation at the promoter and in the coding region of HvBM5
correlate with growth habit in a panel of 10 barley genotypes, (ii) growth habit
correlates with the presence/absence of Vrn-H2 in the same germplasm array, (iii)
allelic architecture at the Vrn-H candidate loci explains QTL mapping results, and (iv)
expression of HvBM5 in representative genotypes, in response to vernalization and
photoperiod treatments, followed patterns predicted from the literature and from the
allelic architecture at the
Vrn-H1
and Vrn-H2 candidate loci.
Structural and Functional Characterization of Candidate Vernalization genes in
Barley
Jarislav von Zitzewitz', Jeffrey S. Skinner1'2, Peter Szücs3, Tony H.H. Chen2, Patrick
M. Hayes1, Jorge Dubcovsky4, Liuling Yan4
1Department of Crop and Soil Science, Oregon State University, Corvallis, OR 97331.
2Department of Horticulture, Oregon State University, Corvallis, OR 97331.
3Agricultural Research Institute of the Hungarian Academy of Sciences, H-2462
Martonvásár, Hungary
4Department of Agronomy and Range Science, University of California Davis, 95616
CA, USA
Plant Physiology
American Society of Plant Biologists
15501 Monona Drive, Rockville, MD 20855-2768, USA
(Submitted)
ABSTRACT
Vernalization, the requirement of a period of low temperature to induce the
transition from a vegetative to a reproductive state, is an evolutionarily and
economically important trait in the Triticeae. The genetic basis of vernalization in
barley (Hordeum vulgare subsp. vulgare) was first defined in terms of a three-locus
epistatic model. We recently cloned candidate genes for two of the vernalization loci
Vrn-A1 and Vrn-A2
in diploid wheat. Here we describe the sequence and expression
of HvBM5A, a candidate for Vrn-H1, in ten barley germplasm accessions. Differences
in vernalization requirement are most likely due to allelic variation at the HvBM5A
promoter and/or intragenic sites rather than in the coding region. Differences in intron
1 are most consistent with growth habit classifications. HvBM5A expression patterns
correlate with growth habit and meristem development; HvBM5A maps to the
predicted position of Vrn-H1 on chromosome 511; and we therefore conclude that
HvBM5A is a strong candidate for Vrn-H1. The presence/absence of the tightly linked
ZCCT-H gene family members on chromosome 4H perfectly correlates with growth
habit. All reported barley vernalization QTL can be explained by a two-locus model
involving Vrn-H1 and Vrn-H2. Facultative genotypes have the Vrn-H2 locus deletion
and signature "winter" habit alleles at HvBM5A. We are currently investigating if the
coincidence of Vrn-H1 with QTL for multiple winter hardiness-related phenotypes is
due to linkage and/or pleiotropy.
10
INTRODUCTION
Vernalization
low temperature
the induction of flowering by exposure to extended periods of
is characteristic of many temperate zone plants, including "winter
growth habit" forms of the Triticeae. The cloning of candidates for the Vrn-A1 and
Vrn-A2 genes in diploid wheat (Triz'icum monococcum) (Yan et al., 2003 and 2004)
and candidates for the Vrnl gene in hexaploid wheat (Triticum aestivum) (Danyluk et
al, 2003; Trevaskis et al, 2003; Murai et al., 2003) has considerably advanced our
understanding of this trait in economically important crop species. The genetics of
vernalization are well-characterized in Arabidopsis thaliana (see review by Henderson
et al., 2003) and it is now clear that different pathways lead to the same end phenotype
in this model species and in members of the Triticeae (Yan et al., 2003 and 2004).
Vernalization requirement is of interest in the Triticeae because of its role in
determining broad adaptation and due to its association with winter hardiness. Winter
hardiness can be defined as the capacity of a genotype to survive the winter (Hayes et
al., 1997). Three principal components of winter hardiness in the Triticeae are low
temperature tolerance, vernalization requirement, and photoperiod sensitivity. The
three phenotypes are usually associated, e.g. "winter varieties" that are low
temperature tolerant usually require vernalization and are photoperiod sensitive.
However, some of the most low temperature-tolerant genotypes are photoperiod
sensitive and don't require vernalization (Karsai et al., 2001). Such varieties are
termed "facultative" because agronomically speaking, they could be fall- or spring-
sown. The variety Dicktoo, which is a model for winter hardiness research in the
11
barley community (Pan et al., 1994; van Zee et aL, 1995; Hayes et al., 1997; Choi et
al., 2000; Mahfoozi et al., 2000; Karsai et al. 2001; Fowler et al., 2001), shows
facultative growth habit: it is cold tolerant, it is sensitive to short photoperiod, and
does not have a vernalization requirement. There is evidence in wheat that maximum
low temperature tolerance is associated with both vernalization and photoperiod
sensitivity (Mahfoozi et al., 2001). Therefore, from both scientific and agronomic
perspectives, it is important to understand the structure and function of Triticeae
vernalization genes.
Barley (Hordeum vulgare subsp. vulgare) is an economically important crop
model for studying the genetics and physiology of vernalization in the Triticeae.
Barley is a self-pollinated diploid, with abundant genetic variation for the components
of winter hardiness, and an ever-expanding set of tools exist for genetic and molecular
analysis (reviewed in Hayes et al., 2003), including multiple mapping populations,
arrayed BAC clones (Yu et al., 2000), a large EST database, and a microarray (Close
et al., 2004).
Takahashi and Yasuda (1971) proposed a three-locus epistatic model for
vernalization in barley in which genotypes that require vernalization (winter types)
have the allelic architecture Shsh2sh2sh3sh3; all other allelic configurations lead to
no vernalization requirement, e.g. "spring" growth habit. Based on wheat: barley
orthology, the Sh loci we now describe using standard Triticeae nomenclature, with an
"H" to indicate the Hordeum genome, as follows: Sh2 = Vrn-H1 (chromosome Sn); Sh
= Vrn-H2 (chromosome 411); and Sh3= Vrn-H3 (chromosome 111). Allelic variants at
12
the Vrn-H3 locus are reported only in barleys from extreme high or low latitudes
(Takahashi and Yasuda, 1971).
A model explaining the Vrn2/Vrnl epistatic interaction in the Triticeae was
proposed by Yan et al. (2003, 2004) based on the positional cloning of Vrn-A1 and
Vrn-A2 candidates in T. monococcum. Per this model, ZCCT1 (the candidate gene for
Vrn2) encodes a dominant repressor of flowering which binds to TmApl (the
candidate for Vrnl), a MADS-box gene. Vernalization down-regulates the expression
of Vrn2 allowing expression of Vrnl. A lack of vernalization requirement occurs in
genotypes with a deletion for Vrn2, regardless of the allele at Vrnl, as well as in
genotypes that do have Vrn2 but that lack a target binding site for the repressor in
Vrnl. A deletion was reported in the promoter region of Vrn-A1 that correlated with
spring vs. winter growth habit. The candidate barley ortholog of Vrn-A1 was first
described by Schmitz et al. (2000) as barley MADS-box 5 (HvBM5), with no
particular descriptive function. In order to standardize nomenclature we will use the
designation HvBM5 in the remainder of this report. The orthology of barley HvBM5
with T. monococcum Api (Vrn-A1) is supported by expression studies (Danyluk et al.
2003; Trevaskis et al. 2003).
It is important to highlight differences in the genetics of vernalization in
Arabidopsis and the Triticeae and to stress that common names do not imply common
functions. The
Triticeae Api candidate for Vrnl is similar to the Arabidopsis
meristem identity gene APETALA1 (APi), but Arabidopsis APi is not directly
involved in vernalization (Yan et al. 2003; Trevaskis et al. 2003; Murai et al., 2003).
13
In Arabidopsis, the vernalization-requirement phenotype is conferred by
a
combination of functional alleles at the FRIGIDA (FRI) and FLOWERING LOCUS C
(FLC) loci (Burn et al., 1993; Koornneef et al., 1994). ZCCTJ , the candidate for Vrn2,
is similar to FLC (as repressor), but belongs to a different family of transcription
factors and contains a putative zinc finger in the first exon and a CCT domain in the
second exon, in common with the Arabidopsis CONSTANS (CO) and CO-like proteins
(Yan et al. 2004). The CCT domain localizes CO in the nucleus (Robson et al. 2001),
the central gene in the mechanism by which daylength controls flowering (Valverde et
al. 2004). The genetics of vernalization in the model plant Arabidopsis is complex and
additional genes will be identified in the Triticeae that are fundamental players in the
vernalization pathway.
The complexity of vernalization and its role in winter hardiness in the Triticeae
led to the use of quantitative trait locus (QTL) analysis tools for trait dissection, and
diploid barley simplified the task. For example, Laurie et al. (1995) reported
vernalization QTL on chromosomes 4H and 5H in the Igri x Triumph (winter x spring)
mapping population and the QTL positions correspond to the predicted locations of
the Vrn-H2 and Vrn-H1 loci. Francia et al. (2003), reported a vernalization QTL on 511
at the predicted position of Vrn-H1 in the Nure x Tremois (winter x spring) mapping
population. QTL for multiple winter hardiness-related traits were mapped in the
Dicktoo x Morex (facultative x spring) population to chromosome 5H at a position
that corresponds with the predicted position of Vrn-H1 (Karsai et al. 1997). The
availability of multiple barley QTL mapping populations segregating for components
14
of winter hardiness provides a unique opportunity to test the Vrnl/Vrn2 model of Yan
et al. (2004) and to further explore the relationships of vernalization, photoperiod
sensitivity, and low temperature tolerance.
In pursuit of our longer-term objective of completely characterizing the
components of winter hardiness in barley, in this study we focused on vernalization
and asked the question: "does the Vrnl/Vrn2 epistatic model proposed by Yan et al.
(2004) for T monococcum apply to barley?" Our specific goals were to determine if
(i) allelic variation at the promoter and in the coding region of HvBM5 correlate with
growth habit in a panel of 10 barley genotypes, (ii) growth habit correlates with the
presence/absence of Vrn-H2 in the same germplasm array, (iii) allelic architecture at
the Vrn-H candidate loci explains QTL mapping results, and (iv) expression of
HvBM5 in representative genotypes, in response to vernalization and photoperiod
treatments, followed patterns predicted from the literature and from the allelic
architecture at the Vrn-H1 and Vrn-H2 candidate loci.
RESULTS
IdentfIcation and mapping of duplicated candidate genes for Vrn-H1 and Vrn-H3
We had previously determined that the barley HvBM5 cDNA (AJ249144;
Schmitz et al., 2000) encodes the probable barley ortholog of T. monococcum APi
(TmAPJ), the candidate gene for Vrn-Ai (Yan et al., 2003). To isolate the genomic
region containing barley HvBM5, high density filters of the H. vulgare cv. Morex
BAC library (Yu et al., 2000) were screened with TmAP1 to identif' clones harboring
15
HvBM5. Six TmAPJ-positive Morex BAC clones were identified and determined to
form two contigs via fingerprinting and Southern hybridization strategies (data not
shown). Contig 1 is composed of BAC clones 460015, 472K16, 631P8, and 597D19,
while contig 2 is composed of BAC clones 315120 and 143P9. A single pass direct
sequence reaction of the 3' end of the HvBM5-like region present on a representative
BAC clone from each contig (clone 631P8 and clone 315120) revealed that the two
clones contain closely related, yet distinct, HvBM5-like genes. The presence of two
HvBM5 genes in barley is in agreement with the genomic blotting results of Schmitz et
al. (2000). We therefore designated the HvBM5 form present on BAC 63 1P8 as
HvBM5A and the form on BAC 315120 as HvBM5B. The HvBM5A form of Morex
(HvBM5A-Mx) is allelic to the cv. Atlas (HvBM5A-At) cDNA (AJ249 144) reported in
Schmitz et al. (2000).
To determine which HvBM5 form, if either, is a candidate for Vrn-H1, we
mapped both forms in the Dicktoo x Morex (DxM) mapping population (GrainGenes:
p/yjepw.usda.gov/ggpages/DxM/). HvBM5A was mapped as a 5' UTRlocalized sized-based polymorphism, while HvBM5B was mapped as an Alul-based
CAPS marker. HvBM5A mapped to the long arm of chromosome 511 and is centered
directly under the DxM winter hardiness QTL cluster (Figure 1). HvBM5A is localized
in BIN 11 (Cooper et al., 2004) as are the vernalization requirement QTL reported for
Nure x Tremois (Francia et al., 2003) and Igri x Triumph (Laurie et al., 1995) barley
mapping populations. This genome location corresponds to the positions of the Vrn-A1
16
and Vrn-D1 loci in T. monococcum and T. aestivum, respectively (Yan et al. 2003;
2004).
ap321
0.00
6.54
'Il
HorB
19.27
HorC
26.12
30.57
iGpi
saflp96
45.54
Ical
58.35
58.39
ABG452
77.08
100.70
5H
4H
IH
IHVBM5B I
HarD
saflpl 64
116.08
iPgd
123.28
BCD265c
0.00
3.17
6.27
9.74
11.99
17.47
20.48
26.42
28.85
39.62
46.44
57.99
60.24
63.66
66.05
74.30
SOLPRO
BCD265a
Dhn6
88.29
saflp67
saflpl76
ap1920
saflp236
ABG472
saflpl59
saflp2o
saflp36
ABG366
mHs
saflp6l
safJp234
HvSnf2 +IZcCT-HI
BMYI
0.00
saflpl29
9.33
saflp22
saflp75
HvAcIl
15.52
18.83
25.46
29.58
saflp2O7
44.81
ABC3O2
61.77
69.62
75.83
76.29
81.24
92.38
mSrh
WG364b
HvCBF9
HvCBF6
saflp58
mR
DsT25
Dhn2
99.81
106.77
108.00
108.12
apADH
saflpl72
IHvBM5A I
BAC635P2
142.62
BCD265b
Dhn9
Ebmac824
saflp2l 8
ABG391
ABG373
153.57
saflpl 04
saflpl 61
161.85
GMSOO1
114.27
118.96
121.94
142.47
apHrth
155.40
163.82
132.11
Figure 1. Linkage map positions of HvBM5A, HvBM5B and inferred position of
ZCCT-H in the Dicktoo x Morex population. The position of ZCCT-H is inferred
based on its tight linkage with SnJ2 (Yan et al., 2004). The positions of candidates for
Vrn-H genes are shown in boxed text. Lines with triangular peaks show the positions
of the vernalization QTL effects attributed to Vrn-H1 and Vrn-H2, with the peak
centered at the most significant position (Laurie et al., 1995; Francia et al., 2003;
Karsai et al. 2004).
17
HvBM5B mapped to chromosome 111, confirming that it is a distinct gene and
not an allele of HvBM5A. The map position of HvBM5B may be coincident with that
predicted for
linkage of
Sh3 (Vrn-H3).
Sh3
We inferred this map position based on the reported
with Bip (Takahashi and Yasuda, 1971) and the barley bin map
(Cooper et al., 2004). The HvBM5B allele in Morex appears to be a 5'-truncated
pseudogene and does not appear to be expressed in Dicktoo, Morex, or Strider (see
below). Relative to the HvBM5A-Mx gene, HvBM5B-Mx only retains the region from
exon 3 through the 3' UTR, while the region 5' to exon 3 is deleted except for
renmants of introns 1 and 2 (Figure 2). We are currently characterizing the structure
and function of HvBM5B alleles in multiple germplasm accessions to establish its
candidacy as
Vrn-H3.
Intron 1 deletion relative to winter and facultative
HvBM5A-Mx
2
HvBMSB-Mx
Hs'BM5A mapping
H
HvBM5A intron 1
primers
i
genotyping primers
Key:
HvBM5B mapping primers
lntron
Exon
HvBM5A/BM5B
RT-PCR primers
UTR
V4
Unknown
...........
Deletion
1.0kb
Figure 2. Exon/intron structure of Morex HvBM5A and HvBM5B, and primer
positions. Arrows indicate positions of genotyping, mapping, and RT-PCR primers.
From left to right, these primer sets are HvBM5.001 and HvBM5.006; HvBM5.055 and
HvBM5.056; HvBM5.032 and HvBM5.035; and HvBM5.004 and HvBM5.070. The
triangle symbol shows the approximate position of the MwoI restriction site used for
distinguishing between HvBM5A and HvBM5B cDNA and gDNA.
18
Coding region variation in HvBM5A alleles does not explain differences in
vernalization requirement
In order to determine if allelic variation at HvBM5A accounts for differences in
the vernalization requirement of representative barley germplasm, we first sequenced
the Morex HvBM5A allele from approximately 2.1 kb upstream of the starting ATG
through approximately 300 bp downstream of the termination codon. The HvBM5A-
Mx genomic DNA sequence (AY750995) was determined via direct sequencing of
both strands of BAC clone 631 P8 across this region. GenBank searches revealed
numerous HvBM5A ESTs from multiple varieties, including Morex. We completely
sequenced one of the Morex HvBM5A ESTs (HVSMEaOO13C13, Accession
BF624256) and confirmed that this cDNA is 100% identical to the coding region and
UTR segments present in the genomic HvBM5A-Mx sequence derived from BAC
631P8. HvBM5A has an intron/exon structure identical to TmAP1 consisting of eight
exons and seven introns. Like TmAPJ, HvBM5A-Mx introns 1 and 2 are large,
consisting of 5590 bp and 2471 bp, respectively.
Primers localized to the 5' and 3' UTRs of HvBM5A-Mx were used to amplify
the corresponding HvBM5A cDNA from the panel of nine additional representative
barley genotypes. These genotypes include eight accessions of Hordeum vulgare
subsp. vulgare that are parents of winter hardiness-related mapping populations: four
winter (Kompolti korai, Nure, Strider, and Igri), two facultative (Dicktoo and
88Ab536), and two spring (Morex and Harrington) genotypes. We also included an
accession of Hordeum vulgare subsp. Spontaneum (OSU 11), the ancestral winter
habit form of cultivated barley. The OSU 11 accession (Caesarea 26-24) is not a
19
mapping population parent but has been extensively genotyped and phenotyped
(Matus and Hayes, 2002; Matus et al., 2003; Karsai et al., 2004). We also included the
variety Trernois, which was used as the spring habit parent in a winter hardiness
mapping population (Francia et al., 2003). However, this variety has been described as
both a winter and spring type in the European Barley Database (http://barley.ipk-
gterslbep.de/ebdb.php3).
HvBM5A coding region alleles were successfully cloned from each genotype
and sequencing of the cDNAs revealed that the products are nearly identical at the
nucleotide level. Only three polymorphic sites are present and are UTR-localized
(Appendix Figure Al). These consist of one 5' UTR-localized 4 bp InDel event and
two single nucleotide polymorphism (SNP) sites: one in the 5' UTR and one in the 3'
UTR. At the two SNP sites, Morex and Harrington (spring genotypes) are identical
and distinct from the other eight genotypes. Morex, Hamngton, and OSU1 1 (winter
habit) have a 5' UTR 4 bp insertion compared to the other winter and facultative
accessions. Tremois (which has an ambiguous growth habit description) has the three
UTR haplotype variations characteristic of the winter and facultative genotypes.
The coding region from each variety is 100% identical at the nucleotide level
and thus specifies an identical HvBM5A polypeptide (data not shown). HvBM5A
encodes
a
MADS-box
protein
that
phylogenetically
clusters
with
the
AP1/SQUAMOSA branch and BLAST searches reveal that it is most closely related,
and likely orthologous to, TmApl and TaVRT-1, in agreement with previous reports
(Yan et al., 2003, Danyluk et al., 2003, and Murai et al., 2003) (data not shown).
20
These results further support the hypothesis that HvBM5A is Vrn-H1 and demonstrate
that variation in the HvBM5A polypeptide does not account for differences in
vernalization requirement observed in this panel of accessions.
The HvBM5A-At polypeptide reported by Schmitz et al. (2000) is from a
spring
variety
(European
Barley
Database:
http://barley.ipk-gatersleben.de
/ebdb.php3) and contains two amino acid substitutions relative to the ten varieties
analyzed in this study (Figure 3). The HvBM5A-At cDNA was isolated as a 5'truncated clone and a full-length eDNA derived via RT-PCR (Schmitz et al., 2000).
These amino acid substitutions may represent actual spring genotype variations not
shared with Morex and Harrington or simply be artifacts resulting from polymerasebased substitutions.
HvBM5A expression correlates with growth habit
The lack of variation among the ten genotypes in the HvBM5A polypeptide
suggests that differences in vernalization requirement in barley may be due to
differences in HvBM5A regulation, as reported by Yan et al. (2003) for TmAPJ of 11
monococcum. We therefore monitored HvBM5A expression in representative spring
(Morex), facultative (Dicktoo), and winter (Strider) barley genotypes under
unvernalized, short day (SD) vernalized, and long day (LD) vernalized conditions
through eight weeks of treatment.
21
NIA 1) S-I
HvBX5A-St
HVBM5A-At
TaVRT-1
ThAp1
LtMADS1
OSMADS14
Z4
AtAP1
PbFBP2 6
AtFUL
HvBM5A-St
HvBN5A-At
TaVRT-1
F
a
a
a
TmAP1
LtMADS1
OsMADS14
I
Zxnm4
a
L
I
iN
I
N
I
N
N
N
N
I
AtAP1
PhFBP26
I
.
a
ALFUL
-
.
A
-I
I
I)
II
C domain
EvBM5A-St
BVBM5A-At
I
PaVRT-1
TmAP1
LtMADS1
OaMADS14
Zmm4
AtAP1
PhFBP2 6
AtFOL
I
RI)AAPVADTSNFIPAAA E:pAEnvAvoIQvpI,RT
ER.EDVAVQPQvPIRTA
RIIAPPAADS I IIPAAA ERAFDAAVQPQAP[RT1
RIIAPPAANS I IIPAAA ERAEDAAVQPAPIRTc
RiIALPU rNLSNYPAAA ER[EDVAAGQPQHARI
L
P
P
P
EAAPJTNVStFPVAA ARVVEGAAAQP-QAV
p
(AA'UPA NASNIPAAA5I)RAEDATG--pp1RTV
NU ---------NU ---------NG---- ------
r: T
NO
NNG
NO
AC ----------
PPQQHQ1QHPYMLSH() PSPFLNNGGLYQEDDPMRNDLF
P
SQPLNSLHWEAYPSALDNGEVEGSSRQQPP
A
PQYCVTSSRDGFVERVGNG- --GASSLTEPNSLJ A
RPTTTNF
LEPVYNCNWCFAA
NG
Figure 3. Alignment of predicted peptides of HvBM5A and other MADS-box
AP1/SQUAMOSA family members. The representative sequence shown is from
cultivar Strider (HvBM5A-St). Also shown are Hordeum vulgare HvBM5A-At
(CAB97352), Triticum aestivum TaVRT-1 (AAP33790), Triticum monococcum
TmAP 1 (AA072630), Lolium temulentum LtM4DS1 (AAD 10625), Oryza sativa
OsMADSJ4 (AAF 19047), Zea mays Zmm4 (CAD23417), Arabidopsis thaliana
AtAP1 (CAA78909), Petunia hybrida PhFBP26 (AAF19164), and Arabidopsis
thaliana AtFUL/AGL8 (Q38876). Areas in black are identical or encode similar
proteins. MADS-box domain = DNA binding domain; I domain intervening region;
K domain = keratin-like domain; C domain = C-terminal region.
At the initiation of the experiment (0 weeks), while HvBM5A expression was
readily detectable in the spring genotype (Morex), no expression was observed in
either the facultative (Dicktoo) or winter (Strider) genotypes (Figure 4A). In the
unvernalized treatments, the HvBM5A expression pattern varied with growth habit:
expression was detectable in Morex at each sampling point, reaching maximum levels
22
by four weeks; expression was detectable in Dicktoo by week two and peaked by
week four; and no expression was detected in Strider over the eight week time course.
With vernalization treatment, the observed HvBM5A expression pattern correlated
with vernalization requirement, while the effect of photoperiod duration (e.g., short
day (SD) vs. long day (LD) varied with genotype. Expression reached maximal levels
in Morex by the first week, and photoperiod did not have a major effect on HvBM5A
expression. There was a low level of HvBM5A expression detectable in Dicktoo
following one week of vernalization, which was slightly higher under LD relative to
SD conditions (Figure 4B). The HvBM5A expression difference between SD and LD
seem to be more evident at two weeks of vernalization; Dicktoo is a photoperiod
sensitive genotype (sensu Karsai et al., 2004). However, even after eight weeks of
vernalization under both photoperiod regimes, HvBM5A expression in Dicktoo was
always lower than in Morex. In contrast, there was no detectable HvBM5A expression
in the winter genotype Strider, under either photoperiod, until eight weeks of
vernalization had elapsed, with the HvBM5A expression level slightly higher under LD
(Figure 4B); Strider is also a photoperiod sensitive genotype. Even after the eight
weeks of vernalization, HvBM5A expression was lower relative to Dicktoo and Morex.
When previously-vernalized Morex, Dicktoo, and Strider plants were placed
back under LD, non-vernalizing conditions, the resultant effect on HvBM5A
expression patterns differed. HvBM5A transcript levels were reduced in Morex, and
more so in plants that had been vernalized under SD conditions. In contrast, no change
in HvBM5A expression was observed for Dicktoo or Strider plants vernalized under
23
SD, while a substantial increase occurred in plants that had been vernalized under LD.
These results highlight the complexity of photoperiod and vernalization interactions in
determining winter, facultative, and spring growth habit; experiments designed to
reveal the nature of these interactions are underway.
We used a diagnostic restriction-profile based reverse transcnptase PCR (RT-
PCR) strategy to verify that the putative HvBM5B pseudogene is not expressed and
therefore not contributing to the observed HvBM5A expression levels. We designed a
primer pair that amplifies an intron-spanning portion of the 3' coding region of both
HvBM5A and HvBM5B, and which yields different size products following MwoI
digestion. This primer pair therefore distinguishes HvBM5A (149 bp) from HvBM5B
(74 and 75 bp) products of cDNA origin, as well as from the larger HvBM5A and
HvBM5B intron-containing products of gDNA origin (Figure 4C). Analysis of RNA
from Morex, Dicktoo, and Strider sampled at time 0 (pretreatment) and at four weeks
of LD-vernalization demonstrated that only the 149 bp HvBM5A cDNA product was
present and there was no evidence for the 74-7 5 bp HvBM5B-derived cDNA product.
Our sequence data, the RT-PCR results, and the lack of HvBM5B-like EST sequences
in GenBank (data not shown), all suggest that HvBM5B is a pseudogene that is not
expressed in any of the three representative genotypes that we studied. The RT-PCR
data paralleled our Northern data for the three genotypes, except that the higher
sensitivity of RT-PCR allowed detection of lower expression levels, e.g., low levels of
HvBM5A expression at time 0 in unvernalized Dicktoo and expression after four
weeks of vernalization in Strider.
24
A
Weeks:
Photoperiod:
0
2
1
LD LD
LD
4
LD
Post
Vernalization
Vernalized
Unvernalized
8
LD
LD
SD
4
2
1
SD
LD
8
LD
SD
SD
2
LD
SD-LD LD-LD
Strider
Dicktoo
Morex
-- -
Strider
at
Dicktoo
.
Morex
I
B
Strider
Dicktoo
c
Unvernalized
Week 0
-
,
.
Vernalized
Week 4
-
.
.
E
HvBM5A cDNA
cDNA
Figure 4. Northern blot of HvBM5A in Strider (winter), Dicktoo (facultative) and
Morex (spring) (A); contrast enhanced images of Strider and Dicktoo (B); and RTPCR of HvBM5A and HvBM5B in Strider, Dicktoo and Morex. (A) Northern blot
probed with Morex HvBM5A cDNA and hybridized with total RNA. Unvernalized
plants were grown under 1 6h light! 8 h dark photoperiod, where week 0 represents the
beginning of the third leaf stage under greenhouse conditions. For vernalization
treatments (2°C) plants were grown at either 8 h light!16 h dark photoperiod (SD) or
16 h/ 8 h dark photoperiod (LD). The last two columns on the blot represent the same
vernalized SD and LD-grown plants that were transferred back to greenhouse
conditions and grown for another 2 weeks under LD photoperiod. 18S rRNA was used
as a Northern hybridization control. (B) Contrast-enhanced image of the HvBM5A
northern analysis for Strider and Dicktoo showing low abundance transcripts and
effects of LD and SD photoperiod. (C) RT-PCR results for Strider, Dicktoo, and
Morex using primers that amplif' a conserved region in HvBM5A and HvBM5B
(Figure 2). HvBM5A cDNA is amplified but there is no amplification of the restriction
digest products of HvBM5B.
25
HvBM5A expression parallels the vegetative to floral meristem transition
The formation of a double-ridge structure on the apical meristem of cereals is a
phenotypic marker of meristem transition from the vegetative to reproductive state
(Danyluk et al., 2003). In conjunction with the tissue harvests for expression analysis,
we investigated the developmental state of the apical meristem at each collection time
point. As shown in Figure 5, the presence of the double ridge structure, indicating the
apical meristem had transitioned to a reproductive state, correlated with HvBM5A
expression. For Morex and Dicktoo, doubk ridge formation was observed at the start
of the experiment, indicating that approximately two and a half weeks of LD growth
under greenhouse conditions, at which time the plants had developed to the two to
three leaf stage, was sufficient to initiate meristem transition in these two genotypes.
The double ridge formation was more advanced in Morex than in Dicktoo at time 0
(Figure 5), indicating Morex had transitioned to a reproductive state earlier than
Dicktoo. This is in agreement with the higher HvBM5A expression level of Morex at
this time point relative to Dicktoo, where expression was only detectable via RT-PCR
(Figures 4A, 4B and 4C.). Strider, which had no detectable HvBM5A expression at this
time point, had not yet initiated double ridge formation. Double ridge formation was
not observed in Strider until eight weeks of vernalization had transpired and is in
agreement with the RNA gel blot analysis, although a low level of expression was
detectable at four weeks of vernalization via RT-PCR.
26
Unvernalized
Weeks: 0
2
Photoperiod:
LD
Vernalized
2
8
SD LD SD LD
Figure 5. Apical meristems of Strider, Dicktoo, and Morex sampled at specific time
points and treatments shown in expression studies (Figure 4). Morex (spring) and
Dicktoo (facultative) show double ridge formation (indicated with a white bracket in
the boxed expanded images) at time point 0 (pretreatment) whereas Strider does not
show double ridge formation until the eighth week of vernalization.
27
For all three genotypes, shoot apex growth was retarded to varying degrees
under low temperature (vernalizing) conditions and may simply be indicative of a
decrease in metabolic activity due to the cold temperature. Strider, however,
essentially ceased growth, whereas some growth was observed in the Dicktoo and
Morex apices. For these two genotypes, growth under low temperature conditions was
more rapid under LD conditions. This may be due to photoperiod regulation of growth
and/or the higher light energy received under LD conditions.
Differences in HvBM5A promoter sequence are generally predictive of growth habit
We sequenced approximately 2.1 kb of upstream regulatory region from each
of the ten barley genotypes to investigate the basis of the HvBM5A expression
differences. Alignment of the promoters revealed that thirty six polymorphic positions,
consisting of SNPs, InDels, and SSRs, were present between the ten genotypes (Figure
A2). Nineteen of the polymorphic positions were specific to an individual genotype,
and are therefore indicative of genotypic variation that is most likely not associated
with growth habit. The remaining seventeen were consistent with spring and winter
growth habit, and the facultative genotypes (Dicktoo and 88Ab536) harbor the winter
haplotype at all positions (Figure 6A). In contrast the promoters of OSU 11 and
Tremois have features of both the winter and spring promoter haplotypes at the
seventeen polymorphic positions. Since OSU 11, a H. vulgare subsp. spontaneum, is
the ancestral winter habit form, the "winter" regions of its promoter may define
regulatory segments that contribute to the winter growth habit. However, analysis of
28
additional accessions of subsp. spontaneum will be necessary to demonstrate this.
Tremois has a unique promoter haplotype that may have originated from
recombination between winter and spring forms.
A
Winter
Facultative
[
osuii [
Tremois
Spring
II
I
I
I
I
III
p
[
I I]
AAP
I
I
I
I
I I
MPA
[
I
I
I
iii
iii
I I]
P CArG
lx CRTIDRE
0 ABRE/G-box
B
Predicted TATA-box
Putative CArC
HvBM5A-Kk
HvBN5A-Nu
HvEM5A-St
HvBM5A- Ig
HvBM5A-Dt
HVBM5A-Ab
HvBM5A-Tr
HvBM5A-011
HvBN5A-Mx
HVBM5A-Ha
Th,API-DV92
ThAPI-G1777
T,nP1-G3 116
TmAPI-G2528A
Th,API-G2528B
ThiAP1-P1349049
ThiAPI-P1355515
Figure 6. HvBM5A promoter (2 kb) haplotypes in winter/facultative and spring
genotypes vs. Tremois and OSU 11 with predicted motifs(A) and predicted TATAbox and the putative CArG motif (B). (A) Genotype-specific polymorphisms were not
included. Conserved "winter" promoter regions are shown in white and conserved
"spring" promoter regions in black. (B) The putative direct/indirect Vrn2 repression
site (CArG motif black arrow on diagram A) detected in T. monococcum (Yan et al.,
2003) is conserved in barley and invariant between winter and spring genotypes.
29
Analysis of the strictly conserved regions between all the genotypes revealed a
number of predicted regulatory motifs that may contribute to common HvBM5A
expression properties (Appendix Figure A2, Table 1). These include multiple light
responsive (data not shown), CArG, and low temperature responsive CRT motifs
(Table 1). Interestingly, the CArG-related motif proposed to be a basis of differential
vernalization requirement between most spring and winter wheat accessions (Yan et
al., 2003), is invariant among all ten of the barley genotypes, demonstrating that this
position is not a candidate for the barley vernalization phenotype (Figure 6B). We
investigated the seventeen polymorphic promoter positions that differentiate winter
from spring genotypes to determine whether any led to a presence/absence event of a
motif between the two growth habit forms. However, no motif with an obvious
function that would explain the winter vs. spring vernalization phenotype was
identified. Overall, the composite structure of the Tremois promoter, along with the
lack of an obvious promoter-based variation that explains the winter vs. spring
vernalization response, suggests that a gene region distinct from the promoter could be
involved in growth habit regulation of HvBM5A expression.
A length polymorphism in HvBM5A intron 1 is correlated with growth habit
Intron-based regulation of MADS-box genes related to flowering and
vernalization has been reported for AG (Sieburth and Meyerowitz, 1997) and FLC (He
et al. 2003; Bastow et al. 2004). Accordingly, we extended our search for
polymorphisms to the introns of HvBM5A. Overlapping amplifications performed
30
along the full length of the Morex and Strider HvBM5A alleles established the
presence of a prominent InDel event (an approximately 5.2 kb deletion in Morex
relative to Strider) that occurs approximately 1 kb into the first intron (Figure 2).
Using primers that flank the borders of this InDel event, we determined that the intron
1 fragment length polymorphism is consistent with the growth habit of the genotypes
(Figure 7). The winter, including OSU1 1, and facultative genotypes, all contain the
longer intron 1 form, whereas the spring genotypes Morex and Harrington have the
truncated form. Tremois, interestingly, contains the longer intron 1 form indicative of
winter and facultative genotypes. A similar finding has been observed for the first
intron of TmAPJ from winter vs. spring wheat accessions, where winters have the
larger intron 1 form (Fu et al., submitted). This 5.2 kb deletion event in spring
genotypes is therefore a strong candidate region for the Vrn-H1 QTL effect source.
Element Class
CArG-like
CArG-like
CArG-like
CRT/DRE
CRT/DRE
CRT/DRE
CRT/DRE
CRT/DRE
ABRE/G-box like
CAAT-box
TATA Box
Sequence
CCAATTTAAG
CCAAAAGTAG
CCTCATTTGG
CCGAC
CCGAC
CCGAC
CCGAC
GTCGAC
CACACGCAGC
CCAAT
TTTAAAA
Function/Response
Developmental/Floral
CC(A/T)6GG
Developmental/Floral
CC(A/T)6GG
Developmental/Floral
CC(AIT)6GG
Abiotic stress (Cold/Drought/Salinity)
CCGAC
Abiotic stress (Cold/Drought/Salinity)
CCGAC
Abiotic stress (Cold/Drought/Salinity)
CCGAC
Abiotic stress (Cold/Drought/Salinity)
CCGAC
Low temperature binding
GTCGAC
Abscisic acid and light response
CACGTG
Basal promoter element
CCAAT
TATA(T/A)A(T/A)A Basal promoter element
Consensus
Table 1. Conserved HvBM5A promoter elements found in ten barley genotypes that
could be involved in vernalization requirement or abiotic stress responses (see Figure
6A for relative positions)
31
Subsequently, we
isolated
and
sequenced HvBM5A
intron
1
from
representative winter (Strider) and facultative (Dicktoo) accessions for comparison
with the spring growth habit allele of Morex. The HvBM5A intron 1 sequence of
Dicktoo (AY750994) and Strider (AY750993) are 100% identical. We report
elsewhere that we have delimited a 2.8 kb deleted region of HvBM5A/TmAP1 intron 1
by comparing overlapping deletions in the Triticeae A, B, D, and H genornes (Fu et
al., submitted). Sequence comparisons revealed that a 436 bp segment of this 2.8 kb
segment display the highest sequence conservation between barley and wheat,
suggesting that conserved regulatory elements important to the winter vernalization
genotype may be present within this region (Figure 7B). Sequence analysis of this 436
bp region indicates a number of predicted cis regulatory motifs are present that may
contribute towards the vernalization phenotype. These include hormone and multiple
light responsive motifs, as well as a cold responsive CRT/DRE motif (Table 2; data
not shown). Most interestingly though, four Dof protein binding sites are also present
in this region and the predicted elements are strictly conserved between wheat and
barley (Figure 7B); Dof proteins function as transcriptional activators or repressors in
plants (Yanagisawa and Sheen, 1998). The candidate Vrn2 proteins ZCCT-Ha
(AAS60249) of barley and ZCCT1 (AY485969) of T. monococcum (Yan et al., 2004)
contain a Dof zinc finger domain, suggesting one or more of these Dof sites may be a
target of Vrn2 repression for HvBM5A and TmAP1, respectively. Experiments to
elucidate which, if any, of these sites are important to the vernalization response of
HvBM5A are planned.
32
Facultative Spring
Winter
Ir
!
100
*
120
*
140
*
160
180
*
200
*
220
*
240
320
HvBM5A-St
TaVRTI-TDC
TrrAP1-DV92
*
HvBM5A-St
TaVRTI-TDC
33*API-DV92
*
GATCAAAGTA
*
260
*
340
280
*
300
*
360
*
380
*
HvBM5A-St
TVRT1-TDC
TmP1-DV92
*
HvBM5A-St
TaVRTI-TDC
TmAPI-DV92
CGGTP
400
I
TrGCTCCT
*
HvBM5A-St
L
i--
420
*
440
ATCTTTGTCT
Figure 7. HvBM5A Intron 1 length polymorphism in winter, facultative, and spring
barley genotypes (A) and sequence alignments of a conserved portion of intron 1 for
HvBM5A, TaVRTJ and TmApl(B). (A) Agrose gel showing that only the spring
genotypes (Barrington and Morex) contain an approximately 5.2 kb deletion. (B)
Sequence alignments for a 436bp region within the 5.2 kb fragment that is highly
conserved between H. vulgare subsp. vulgare (HvBM5A), T. aestivum (TaVRTJ) and
T. monococcum (TmAPJ). Underlined conserved sequences below the alignment
indicate positions of putative Dof binding sites.
33
Element Class
I-box like
C-box like
RY motif
ABRE/G-box like
CRTjDRE
MRE
MRE
TATC-box
Dof
Dof
Dof
Dof
Sequence
GTTAT (-)
CGTCA (-)
CATGCA
ATCACGTGAC
CCGAC
AACCAAA
AACCTAT
TATCTCA
GATCAAAGTAT
AAATAAAGCCG
AGGCAAAGCAA
AGACAAAGCAT
Consensus
Function/Response
GATAA
TGACG
CATGCA
CACGTG
CCGAC
AACCTAA
AACCTAA
TATCCCA
NMNNAAAGNNN
NNNWAAAGCNN
NNNWAAAGCNN
NNNWAAAGCNN
Light responsive
MeJa-responsive, light responsive
ABA responsive, seed expression
abscisic acid responsive, light responsive
Abiotic (cold, drought, salt) stress inducible
MYB transcription factor binding site
MYB transcription factor binding site
Gibberellin responsiveness
Dof transcription factor binding site
Dof transcription factor binding site
Dof transcription factor binding site
Dof transcription factor binding site
Table 2. HvBM5A intron 1 elements found in a 436 bp region conserved between
Hordeum and Triticum accessions, involved in flowering or abiotic stress responses.
The presence/absence of Vrn-H2 correlates with vernalization requirement and
defines facultative growth habit
Vernalization loci interact in an epistatic fashion, where the vernalization
requirement phenotype is observed only in Vrn-H2
vrn-Hlvrn-Hlvrn-H3vrn-H3
genotypes (Takahashi and Yasuda, 1971; Laurie et al., 1995; Yan et al., 2004). As
noted above, we hypothesize that Vrn-H3 alleles in our germplasm array are nonfunctional, reducing the genetic model from three to two interacting loci. Consistent
with the hypothesis that Vrn2 encodes a repressor of Vrn], we previously observed
complete deletions of the ZCCT-H candidate genes for the Vrn-H2 locus in spring
barley germplasm accessions Yan et al. (2004). We extended the Vrn-H2 locus
germplasm characterization to the ten genotypes examined in this study by multiplex
amplification of a conserved region of the Vrn-H2 candidate genes ZCCT-Ha and -Hb
(referred to as ZCCT-H in the remainder of this report), as well as the tightly-linked
34
Vrn-H2
locus-proximal
amplifications, the
SnJ2
Vrn-H2
gene (Yan et al., 2004). Based on the ZCCT-H gene
locus is deleted in both the facultative and spring
genotypes, being present only in genotypes that display a strong vernalization
response (Figure 8). In contrast,
Vrn-H2
locus-proximal
varieties (Figure 8), verifying that the lack of
SnJ2
ZCCT-HaIb
amplified from all ten
amplification in the
facultative and spring genotypes is likely due to a physical deletion of these genes and
not a failure of PCR amplification. These results provide a simple genetic model that
explains the facultative growth habit: The physical deletion of the
results in loss of the trans-acting repressor of the winter
Vrn-H1
Vrn-H2 locus
allele, yielding a
pseudo-spring, facultative genotype from which a vernalization requirement has
essentially been eliminated. The lack of a strong vernalization response for Tremois,
despite containing an apparent winter
deletion of
Vrn-H2
Vrn-H1
intergenic allele, is likely due to
and suggests that Tremois may actually be better classified as a
facultative genotype.
Facultative
Winter
E
a
z
rID
0
Spring
-
ZCCT-H
SnJ2
Figure 8. PCR amplification of SnJ2 and ZCCT-H showing the deletion of the latter in
facultative and spring genotypes.
35
The Vrn-H1 and Vrn-H2 epistatic interaction model agrees with QTL and Mendelian
mapping results
The Vrn-H2/ Vrn-H1 epistatic interaction model, based on the presence/absence
of Vrn-H2 and the promoter and/or intron 1 architecture at the Vrn-H1, explains the
vernalization requirement QTL results reported in every barley mapping population
reported in the literature (Table 3) except for Nure x Tremois and Igri x Triumph.
According to the initial report of Francia et al. (2003), the Nure x Tremois population
showed a vernalization QTL only on 5H, in association with Vrn-H1. This QTL effect
may be attributable to the "winter" Vrn-H1 allele in Nure and the unique Vrn-H1 allele
in Tremois. No QTL were reported on 4H in association with Vrn-H2, but this was due
to an incomplete genetic linkage map in this region. We therefore scored additional
loci in this population, which expanded the linkage map to include all the long arm of
4H (Figure: data provided after completion of thesis). Using this linkage map and the
available phenotype data, we now have strong evidence for a QTL on chromosome
4H. A controlled environment assessment of vernalization requirement and
photoperiod sensitivity in the Nure x Tremois population is underway. The
significance of QTL in association with both Vrn-H1 and Vrn-H2, as reported by
Laurie et a! (1995) in Igri x Triumph, would be predicted if alleles are segregating at
both loci. We genotyped Igri in this study and confirmed it has winter alleles at both
loci: unfortunately, we were not able to obtain seed of the Triumph mapping
population parent to confirm its allele architecture at the Vrn-H2 and Vrn-H1 loci.
Further support for the two-locus epistatic model comes from the vernalization
phenotype data from the progeny of a Morex x Hordeum vulgare subsp. spontaneum,
accession OSU6 cross. We present these data in greater detail elsewhere (Fu et al.
submitted). Briefly, we have shown Morex is Vrn-H1 Vrn-Hlvrn-H2vrn-H2 and the
spontaneum accession is inferred to be (vrn-Hlvrn-H1 Vrn-H2 Vrn-H2) due its
vernalization requirement (Karsai et al., 2004). The two-way factorial analysis of
variance showed a significant interaction between Vrn-H1 and Vrn-H2 (P= 0.01).
Therefore, we first analyzed the effect of the Vrn-H1 intron 1 deletion (e.g. Morex
allele) within the two Vrn-H2 classes. Among the plants carrying the recessive vrn-H2
(e.g. Morex allele) allele no significant effect on flowering time was observed between
the plants with and without the Vrn-H1 intron one deletion (P = 0.69). However,
among the plants carrying the dominant Vrn-H2 allele (e.g. spontaneum allele), plants
with at least one copy of the Vrn-H1 intron 1 deletion flowered 35 days earlier than
the plants without the deletion (P < 0.0001). Similar results were found when we
analyzed the effect of the Vrn-H2 gene deletion within the two Vrn-H1 classes. No
significant effects for the Vrn-H2 alleles were observed among the plants carrying at
least one Vrn-H1 copy with the intron deletion (P = 0.54). However, among the plants
with the recessive vrn-H1 allele those carrying the recessive vrn-H2 allele flowered 34
days earlier than those with the dominant Vrn-H2 allele (P = 0.0003).
37
Mapping population and
Parents
Vrn-H2
Dicktoo x Morex
Strider x
88Ab536
Nure x Tremois
Dicktoo x
Kompolti korai
Igri x Triumph
Chromosome 5H
Chromosome 4H
Dicktoo
Morex
Strider
88Ab536
Nure
Tremois
Dicktoo
Kompolti
Igri
Triumph
QTL
-
+
+
+
+
-
+
+
+
+
Vrn-H1
Vrn -Hi
promoter
W
intron 1
W
S
S
W
W
W
W
W
W
W
W
W
W
WIS
W
W
W
QTL
+
+
?
Table 3. Summary of Vrn-H1 and Vrn-H2 allele types compared to vernalization
requirement QTL reported in the literature. The presence or absence of Vrn-H2 is
indicated by "+" or "-". VrnH-1 alleles are classified as "winter (W)" or "spring (S)"
based on promoter and intron 1 features. Significant QTL effects are shown as "+";"indicates to QTL effects reported.
*The Tremois promoter has winter and spring features
**The Vrn-H1 and VrnH-2 alleles of Triumph were not sequenced.
DISCUSSION
HvBM5A is a strong candidate for Vrn-H1
Four lines of evidence
orthology, map location, expression pattern during
plant development, and Mendelian genetic analysis of the vernalization phenotype -
provide very strong support for the hypothesis that HvBM5A is Vrn-Hi. Blast
searches reveal that HvBM5A protein sequences of all ten genotypes show highest
identity with TmAPJ and Ta VR T-1 , which are reported to encode a MADS-box protein
(Yan et al., 2003, Danyluk et al., 2003, and Murai et al., 2003). HvBM5A maps to Bin
38
11 of chromosome 5H, which is syntenous with the reported position of Vrn-A1 and
flanking markers in T. monococcum,
and
based on Triticeae homoeology, the
position of Vrnl in the B and D genomes. Bin 11 is also the position of vernalization
response QTL in the Nure X Tremois (Francia et al., 2003) and Igri x Franka (Laurie
et al., 1995) mapping populations. The expression patterns of HvBM5A
in terms of
both amount of HvBM5A niRNA detected and correlation of HvBM5A expression with
meristem differentiation
agree with previous reports in the literature for the putative
Triticum spp. orthologs of Vrn] (Yan et al. 2003; Danyluk et al., 2003. Finally, our
vernalization requirement phenotype data from the cross of Morex x Hordeum vulgare
subsp. spontaneum (OSU6) show an inheritance pattern that agrees with expectations
for a two-locus, epistatic model.
An additional line of evidence that HvBM5A is Vrn-H1 would be complete
association of winter and spring HvBM5A alleles with vernalization requirement in
QTL mapping populations where significant QTL effects are detected on chromosome
5H. As shown in Table 3, the appropriate populations for such an analysis are Nure X
Tremois (Francia et al., 2003) and Igri x Triumph (Laurie et al., 1995). In the Nure x
Tremois population, vernalization requirement was calculated as the difference in
heading date of each map-line in fall spring-sown experiments and is therefore
confounded with earliness per se and photoperiod sensitivity effects. Accordingly, we
are currently characterizing the Nure x Tremois population for vernalization
requirement and photoperiod sensitivity under controlled environment conditions. It is
not possible to determine the Vrn-H1/H-2 allele genotype relationships with
39
vernalization requirement in the Igri x Triumph population based on the published
report (Laurie et al., 1995) because genotype and phenotype data sets are not
available.
Definitive proof that HvBM5A is Vrn-H1 will require forward and/or reverse
genetics strategies. The overall recalcitrance of barley to transformation has slowed
efforts to engineer appropriate genotypes (e.g. with complete Vrn-H2 but with a spring
Vrn-H1) with winter type Vrn-H1 alleles. However, recent progress in tissue culture
promises to remove this impediment (Chang et al., 2003). Yan et al. (2004) used a
gene silencing strategy to demonstrate that ZCCT-1 is Vrn-A2, and this approach may
also be useful for demonstrating the candidacy of Vrn-H1, although selectively
regulating a MADS-box gene that may function throughout plant growth and
development could be a challenge.
Dfferences in vernalization requirement, f due to HvBM5A, are regulatory and not
structural
Given that all the data support the hypothesis that HvBM5A is Vrn-H1, we
asked the question "are differences in vernalization requirement due to structural
and/or regulatory differences in Vrn-H1 alleles?" Our data provide very strong
evidence that differences in vernalization requirement are due to gene regulation, not
in the encoded polypeptide. The coding regions of all ten genotypes we surveyed
which represent an ancestral form and cultivated genotypes from the major germplasm
groups (e.g. spring and winter habit; two-row and six-row inflorescence) of North
America and Europe
are identical. There are limited polymorphisms in the UTRs but
these would not lead to differences in the encoded polypeptide. Integrating these
results with those from Triticum spp. (Yan et al., 2004; Danyluk et al., 2003) reveals a
high degree of Vrn] gene sequence conservation, and presumably function, in the
Triticeae.
Based on the lack of polypeptide variation and the report of Yan et al. (2003)
that promoter mutations in a CArG-like motif accounted for differences in growth
habit in T monococcum, we searched for promoter differences in the ten
representative barley genotypes. We found the CArG-like motif in all ten accessions is
invariant. Furthermore, the T. monococcum CArG-!ike motif is a variant of the
consensus CC(AIT)6GG (Shiraishi et al., 1993; Mizukami et al., 1996), as it has a
sequence of CCTcgTTTTGG, where a C and a G are present in the normally AlT
specific intervening sequence, and the flanking CC-GG are spaced by 7 bp, rather than
the normal 6 bp. The corresponding CArG-like motif of barley has the sequence
CCTCATTTGG and is a better match to the consensus sequence in spacing and
sequence, although it does contain a C in the normally AlT-exclusive 6 bp run
position. Based on these results, this CArG-like motif is not a candidate for winter vs.
spring phenotypes.
Yan et al. (2004) also found dominant alleles of VrnI that do not have CArG-
like motif mutations, and these findings prompted a more comprehensive analysis of
the promoter and intron
1
regions in the A, B, D, and H genomes (Fu et al.,
submitted). Based on the reports of intron-based regulation of MADS-box genes
related to flowering in Arabidopsis (Sieburth and Meyerowitz, 1997; He et al. 2003;
41
Bastow et al. 2004) we characterized the overall structure of intron 1 in the ten
genotypes and
sequenced the full intron in three genotypes representing spring
(Morex), facultative (Dicktoo), and winter (Strider) growth habit classes. We found
that all the winter and facultative genotypes, and OSU 11 (the Hordeum vulgare
subsp. spontaneum accession) , contain a HvBM5A intron 1 that is 5.2 kb longer than
in the spring genotypes Morex and Harrington. Since winter habit is the ancestral form
in the Triticeae, the deletion of this region of intron 1 may be of functional and
evolutionary significance.
As reported elsewhere in greater detail ( Fu et al.,
submitted) we have further reduced the critical region of this deletion to 2.8 kb region.
The first 436-bp of this 2.8-kb region showed the highest sequence conservation
between the A, B, D, and H genomes suggesting that it may be the site of conserved
regulatory elements involved in regulation of growth habit. However, biochemical
andlor transgenic experiments will be necessary to determine if these are indeed the
vernalization response mediating regulatory regions. Considering the Triticeae as a
whole, the data suggest that the regulation of Vrnl may be achieved through the
promoter and/or intronic regions. The mechanisms have yet to be elucidated, and the
question remains if Vrn2 is the direct DNA binding trans-acting regulatory factor of
Vrnl or if Vrn2 is a cofactor in a complex that effects the DNA binding of Vrnl.
The Vrn-H2 deletion explains spring and facultative growth habit
Spring and winter growth habit can be easily defined based on the presence or
absence of a vernalization requirement, and the two-locus epistatic interaction model
42
of Yan et al. (2004) provides a genetic mechanism to explain the phenotype. We found
a complete association of the Vrn-H2 deletion with spring growth habit, extending the
results of Yan et al. (2004) to North American, European and ancestral germplasm
accessions. We also observed a deletion in Dicktoo and 88Ab536, facultative
genotypes, and Tremois, a genotype with conflicting growth habit descriptors. The
term "facultative" growth habit has lacked a rigorous definition, and is used rather
loosely to define genotypes that do not have a vernalization requirement but do have
sufficient low temperature tolerance for fall-sowing in some environments. Additional
confirmation in broader range of germplasm and elucidation the underlying genetic
mechanisms will be necessary, but we propose that the term facultative growth habit
may be appropriate to describe of genotypes that are low temperature-tolerant and
have "winter" alleles at Vrn-H1, but lack the repressor encoded by Vrn-H2. By this
definition, Tremois could be re-classified as a facultative. II meets the Vrn allele
architecture criteria and we are currently characterizing its low temperature tolerance
and other winter hardiness related phenotypes in controlled environment tests. As
spring genotypes typically transition to a floral fate faster than facultative genotypes,
additional regulatory inputs are likely differentially affecting the Vrn-H1 spring alleles
relative to the winter Vrn-H1 allele present in facultative genotypes, and this may
account, in part for differences in maturity.
43
The Vrn2/VrnI two-locus model provides a genetic explanation for vernalization
requirement QTL
Vernalization requirement in barley is not absolute: in a study of a diverse
array of barley germplasm, Karsai et al. (2001) found that when unvernalized, all
genotypes surveyed would eventually flower. However, the heading date of some
genotypes was so delayed when unvernalized as compared to vernalized (e.g. up to
141 days) that these genotypes can be said to have a vernalization requirement.
Accordingly, vernalization requirement/response in barley is measured quantitatively
(e.g. as the difference in heading date between vernalized and unvernalized plants
grown at a specified photoperiod) and these phenotypic data are analyzed using QTL,
as opposed to Mendelian tools. Vernalization requirement QTL are reported from a
number of barley mapping populations, and Vrn loci were hypothesized to be
candidate genes for these QTL effects (Hayes et al., 1997). Our characterization of the
Vrn-H1 and Vrn-H2 locus genotypes in mapping population parents explains QTL
effects in all reported mapping populations (Table 3 ). The first cross combination
listed Dicktoo x Morex is a facultative x spring cross in that both parents show the
Vrn-H2 deletion but have contrasting alleles at Vrn-H1. No vernalization requirement
QTL are expected, due to the lack of the Vrn-H2 repressor, and none are reported.
Strider x 88Ab536 and Dicktoo x Kompolti korai are winter x facultative crosses, and
QTL effects would be expected only in association with Vrn-H2 since the parents
contrast for the deletion but both have winter type alleles at Vrn-H1.
These
expectations are supported by the QTL data (Marquez-Cedillo et al., submitted; Karsai
et al., submitted). By expanding the map of 4H in Nure x Trernois, we have shown
44
that there is a vernalization QTL in this population, as would be expected based on
segregation of the Vrn-H2 deletion. In the case of Igri x Triumph, we had to infer that
Triumph is lacking Vrn-H since we were not able to obtain seed of the Triumph source
used as the parent of the mapping population.
The finding that our Vrn-Hl and Vrn-H2 allele data are sufficient to explain all
reported vernalization requirement QTL data are additional evidence that Vrn-H3 may
be a determinant of vernalization response only in a limited range of barley
germplasm, as reported by Takahashi and Yasuda (1971). We provide preliminary
data that a second form of HvBM5 (HvBM5B) is a candidate for Vrn-H3, although
additional experiments are clearly necessary. The pseudogene structure of HvBM5B,
and the lack of expression of this gene in Strider, Dicktoo, and Morex support the
QTL data.
Vrn loci and other winter hardiness phenotypes
This characterization of the structure and function of Vrn genes in barley is
part of a long-term effort to unravel the complexities of the winter hardiness regulon
in barley that involves a systematic genetic and physiological dissection of key
phenotypes, including vernalization requirement, photoperiod sensitivity, and low
temperature tolerance. This long-term goal has relied heavily on QTL approaches,
given the complex inheritance and interactions of the three phenotypes and, until
recently, a lack of candidate genes. Of particular interest in the current context is the
coincidence of QTL for multiple winter hardiness-related traits on chromosome 5H, in
45
conjunction with the Vrn-H1 locus, in multiple barley mapping populations (Hayes et
al., 2003). This same region has also been implicated in Triticum spp., where the low
temperature tolerance effects in conjunction with Vrnl have been attributed to a locus
designated Fri (see Crosatti, 2003). We are currently investigating the question of
whether these multiple QTL are effects of separate loci (e.g. a cluster of adaptivelyrelated genes) or if they are the pleiotropic effects of the Vrn-H1 locus.
MATERIALS AND METHODS
Plant material and growth conditions
Ten Hordeum vulgare subsp. vulgare varieties representing the three barley
winter hardiness phenotype classes - winter, facultative winter, and spring- were
utilized in the current study. These are the five winter class varieties: Kompolti korai,
Nure, OSU1 1, Strider, and Igri; the two facultative winter class varieties: Dicktoo and
88Ab536; and the three spring class varieties: Morex, Tremois, and Harrington. Plants
for DNA extraction were grown under greenhouse conditions and supplemented with
artificial light (16 h photoperiod; 18°C/15°C day/night temperatures). For expression
studies, Strider, Dicktoo, and Morex varieties were grown to the beginning of the third
leaf stage under greenhouse conditions (16 h photoperiod) and then transferred to
treatment conditions
unvernalized, short day (SD-) vernalized, or long day (LD-)
vernalized and bulk aerial tissue collected following 1, 2, 4, and 8 weeks of treatment
exposure. Prior to transfer to treatment conditions, a pre-transfer (0 exposure) sample
was collected as above from each variety. For the unvernalized treatment, plants were
46
maintained under greenhouse conditions. For the SD- and LD-vernalized treatments,
plants were transferred to environmentally-controlled cold rooms maintained at 2°C
with either an 8 h photoperiod (SD) or 16 h photoperiod (LD). For post-vernalization
treatments, following the 8 weeks of vernalization under either SD or LD photopenod,
plants were returned to the above pretreatment greenhouse conditions (LD) and
samples collected after 2 weeks additional growth. Plants for HvBM5A cDNA
isolations were grown and SD-vernalized as above for 4 weeks prior to tissue harvest.
Isolation of the candidate Vrn-H1 Gene alleles
High density filters of the H. vulgare cv. Morex BAC library (Yu et al., 2000)
were screened with TmAPJ and contig sets determined as previously described (Yan et
al., 2003). High quality BAC DNA for direct sequencing was prepared using a
QIAGEN Large-Construct Kit (Qiagen, Valencia, CA). The sequence of the fulllength Morex HvBM5A gene allele (AY750995) was determined from both strands of
BAC clone 631P8. To determine the HvBM5A coding region sequence for each barley
variety, first-strand cDNA was prepared from 5 jig of 4 week SD-vernalized total
RNA of each respective variety using a RETROscript Kit (Ambion, Austin, TX)
according to the manufacturers instructions. The coding and flanking regions for each
HvBM5A cDNA were PCR-amplified from the respective first strand eDNA pool
using the 5' and 3' UTR-localized primers HvBM5.00 1 (5'-ccaaccacctgacagccatg-3')
and HvBM5.002 (5'-cgataggttaattcacagaaacaac-3') and Qiagen Taq DNA polymerase
according to the manufacturers instructions (Qiagen, Valencia, CA). To determine
47
each respective HvBM5A promoter sequence, approximately 2.1 kb of sequence
upstream of the starting ATG, based on the determined Morex allele present on BAC
clone 631P8, was amplified and cloned from each remaining barley variety. Total
genomic DNA was extracted from each barley variety using a DNeasy Plant Mini Kit
(Qiagen, Valencia, CA) and the promoter region subsequently amplified using primers
HvBM5.027 (5'-aggcctattcgtttgcaatgc-3') and HvBM5.006 (5'-atctcgtgcgccttcttgag-3 )
and Takara LA Taq DNA polymerase (Takara Minis Bio, Madison, WI) according to
the manufacturer's guidelines. For HvBM5B, we used
the following
primers:
and
HvBM5.032
(5'-tcggaaatgaacaaaagagactt-3')
mapping
HvBM5.03 5
(5'-
gaaaacttgaacaacaccagaacc-3'). These primers amplify a 487bp fragment in both,
Dicktoo and Morex. We sequenced both and it mapped the polymorphism as a cleaved
amplified polymorphic site (CAPS) marker (see mapping section). We cloned and
sequenced another region of HvBM5B with one PCR nm of HvBM5.003 (5'gcacgaatccatttctgagcttc-3') and HvBM5.002 (as described in the eDNA section). This
amplicon spans the region from exon 5 to the 3'UTR (see figure 2 for exon position).
Sequences were obtained for this region from 88Ab536, Morex, and Strider, but for
Dicktoo
we
obtained
only
the
3'UTR
with
primers
HvBM5.005
(5'-
cttccagcccatgtaagcgt-3') and HvBM5.002. the RT-PCR primers were designed from an
alignment with of all HvBM5B sequences. For all sequenced PCR products, PCR
amplicons were electrophoretically separated in agarose gels, excised and purified
using QlAquick Gel Extraction Kits (Qiagen, Valencia, CA), and cloned for
sequencing using the TOPO TA Cloning Kit (Invitrogen, Carlsbad, CA). For each
48
gene/allele isolated via PCR, cloned amplicons from two independent PCR reactions
were each sequenced and verified to contain identical sequences to verify polymerase-
based nucleotide substitutions had not occurred; only the non-primer portion of each
sequenced amplicon was reported to GenBank. Regulatory motif searches of the
promoter and barley:wheat conserved intron 1 region were conducted using PLACE
(http://www.dna.affrc.j.o.jp/PLACE/),
MOTIF
(http://inotgrioiiejp/),
and
PIantCARE (http://intra.psb.ugent.be:8080/ P1antCAREI) sequence analysis functions.
Linkage Mapping
Vrn-H loci linkage map positions corresponding to HvBM5A (Vrn-H1),
HvBM5B (Vrn-H3), and Vrn-H2 were assigned using the Dicktoo x Morex (DxM)
doubled haploid mapping population (Hayes et al., 1997). HvBM5A was mapped as an
amplified differential sized-based polymorphism (221 bp-Dicktoo vs. 225 bp-Morex)
present in the 5' UTR using the primers HvBM5.006 (sequence in gene isolation
section) and HvBM5.001 (sequence in gene isolation section). HvBM5B was mapped
as
an Alul-based CAPS marker using the primers HvBM5.032 (5'-tcggaaat
gaacaaaagagactt-3') and HvBM5.03 5 (5'-gaaaacttgaacaacaccagaacc-3'). The candidate
genes for the Vrn-H2 locus (Yan et al., 2004) of barley are physically deleted in both
mapping population parental genotypes (see Results). SnJ2, the closest physicallylinked gene, was used to position the adjacent Vrn-H2 deletions via an approximately
250
bp
differential-sized
amplicon
using
the
primers
cccggattggtcagagaagt-3') and SnJ2.002 (5'-gcggttttagttttgattat-3').
SnJ2.00l
(5'-
based on the
49
methodology of Yan et al. (2002). All mapping-based PCR reactions were performed
using Taq DNA polymerase under standard conditions optimized for each gene and
primer set according to the manufacturers instructions (Qiagen, Valencia, CA).
JoinMap 3.0 (Van Ooijen and Voorrips, 2001) was used for linkage map construction
following procedures by Castro et al. (2002). The linkage map relationship of the Vrn-
Hi candidate gene HvBM5A to the Dicktoo x Morex vernalization QTL position
present on 5H-L was performed using existing phenotype data sets (GrainGenes) and
QTL Cartographer Version 1.6 (http://statgen.ncsu.edulqticart/) on the newly
constructed map.
Alignments and Phylogenetic Analysis
Sequences used for promoter alignments were barley alleles HvBM5A -Kk, HvBM5A
011, HvBM5A -St, HvBM5A -Ig, HvBM5A -Dt, HvBM5A -Tr, HvBM5A -Ab, HvBM5A
-Mx, HvBM5A -Ha, and wheat TmApl alleles reported by Yan et al. (2003). Sequences
used for protein alignments a were full-length proteins for HvBM5A-At (CAB97352),
HvBM5A-St (AY750993), TaVRT-1 (AAP33790), TmAP1 (AA072630), LtMADS1
(AAD 10625), OsMADS 14 (AAF 19047), Zmm4 (CAD234 17), AtAP 1 (CAA78909),
PhFBP26 (AAF19164), and AtFUL (Q38876). For the promoter and protein
alignments, sequences were aligned using ClustalW (http://clustalw.genome.jp/) and
the output alignments refined by hand using GeneDoc Version 2.6 software
(http://www.psc.edu/biomed/genedoc). Phylogenetic analyses on refined alignments
50
were conducted using MEGA Version 2.1 software (http://www.megasoftware.net/;
Kumar et al. 2001).
RNA gel blot and RT-PCR expression analysis
Total RNA was isolated from all tissue sources using RNeasy Plant Kits
(Qiagen, Valencia, CA). Gel electrophoresis and blotting of RNA to Nytran nylon
membranes (Schleicher and Schuell, Keene, NH) was performed as previously
described (Skinner and Timko, 1998). RNA blots were probed and washed using
Ultrahyb Solution following the manufacturer's guidelines (Ambion, Austin, TX). For
northern analysis, an HvBM5A probe fragment was isolated from the Morex cDNA
present in EST HVSMEaOO13C13 that excluded the conserved MADS-box domain.
An 18S rRNA fragment from Morex EST BF620814 was utilized as a constitutive
loading control probe. Labeled probes were generated using a High Prime Labeling
Kit according to the manufacturer's protocol (Roche Molecular Biochemicals,
Indianapolis, N). Diagnostic restriction-profile based RT-PCR was employed to
determine whether HvBM5B was expressed. Dicktoo, Strider, and Morex pretreatment
(0 exposure) and 4 week LD-vernalized RNA was used as a template for cDNA
synthesis. First-strand cDNA for each sample was synthesized from 5 tg total RNA
utilizing a RETROscript Kit (Ambion, Austin, TX) according to the manufacturers
instructions. Input first-strand cDNA sample volumes for RT-PCR were first
normalized for cDNA yield between samples relative to a barley actin gene using the
primers
HvActin.001
(5'-tcgcaacttagaagcacttccg-3')
and
HvActin.002
(5'-
51
aagtacagtgtctggattggaggg-3'). For RT-PCR amplifications, PCR was performed on
each cDNA sample with Qiagen Taq polymerase according to manufacturer's
guidelines (Qiagen, Valencia, CA) for 35 cycles (90°C for 30 s, 55°C for 45 s, and
72°C for 60 s per cycle) followed by a 10 mm final extension at 72°C. Primers
HvBM5.004 (5'-ggcgcagcaagatcaaactca-3') and HvBM5. 070 (5'-ggacctgaggctgcactgc3') were used for HvBM5AIB analysis and designed to coamplify an analogous intron-
spanning 3' portion of the coding region of HvBM5A and HvBM5B that would yield
differential-sized products following MwoI digestion and simultaneously distinguish
products originating from cDNA vs. gDNA templates. To verify both HvBM5A and
HvBM5B amplification would occur under the experimental conditions and that MwoI
digestion of HvBM5B product had taken place, gDNA from Dicktoo, Strider, and
Morex were also used as templates. Following amplifications, reaction products were
digested with MwoI (New England BioLabs, Beverly, MA) and electrophoretically
separated.
Apical meristem development
Shoot apical meristem development was determined in conjunction with the
tissue harvest for the 0, 1, 2, 4, and 8 week expression analysis stages for the
unvernalized, and SD- and LD-vernalized treatments from Dicktoo, Strider, and
Morex; the 2 week post-vernalization treatment time point was not examined. For each
time point, the apical meristems of three individual plants from each barley genotype
were dissected from the crown tissue and digitally captured; an average representative
52
meristem from each relevant time point is presented. Meristem images were captured
using a Nikon Coolpix 995 digital system and a Nikon SMZ-2T dissecting scope.
Meristem developmental stage was determined by the degree of double-ridge
formation as in Danyluk et al., (2003).
Genotyping of HvBM5A intron 1 size and Vrn-H2 candidate gene presence
The ten barley lines were genotyped for HvBM5A intron 1 size and Vrn-H2
candidate gene presence using gDNA as amplification template. To genotype the 5 kb
size polymorphism of the HvBM5A intron 1, PCR amplifications were performed
using Takara LA Taq DNA polymerase (Takara Minis Bio, Madison WI) according to
the
manufacturer's
guidelines
atgcatagaataattggctccagc-3')
and
with
the
HvBM5.056
primers
HvBM5.055
(5'-
(5'-cagtaagcactacgatgatgataaac-3')
followed by electrophoretic separation of the products. Primers HvZCCT.00 1 (5'cacatgatgtcgcccgttc-3') and HvZCCT.002 (5'-ggactcgtagcggatttgc-3') were designed to
coamplify a conserved region of two closely related candidate Vrn-H2 genes, ZCCT-
Ha (AY485977) and ZCCT-Hb (AY485978), and were utilized in a multiplex PCR
reaction with primers HvSnJ2.0 1 F (5'-cctgaagcgagtatccatatgc-3') and HvSnJ2.03R (5'-
gctgattgtttttgtggccagg-3') to simultaneously amplify the Vrn-H2 locus-proximal SnJ2
gene. The Vrn-H2 (ZCCT-Ha/ZCCT-Hb)/Snj2 multiplex PCR reactions were
performed using Qiagen Taq DNA polymerase according to the manufacturer's
instructions (Qiagen, Vanecia, CA) and the products electrophoretically separated.
53
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57
General Conclusions
HvBM5A is a strong candidate for Vrn-H1
There are four lines of evidence that make HvBM5A a strong candidate for
Vrn-H1: orthology, map location, expression pattern during plant development, and
Mendelian genetic analysis of the vernalization phenotype. Blast searches reveal that
the HvBM5A protein sequences is a MADS-box gene showing highest identity with
TmAP] and TaVRT-] (Yan et al., 2003, Danyluk et al., 2003, and Murai et al., 2003).
HvBM5A maps to chromosome 5H (Bin 11), the same syntenous Vrn-A1 region in T
inonococcum, and the position of Vrnl in the B and D genomes based on Triticeae
homoeology. Bin 11 is also the position of vernalization response QTL in other barley
mapping populations (Francia et al., 2003; Laurie et al., 1995). The expression
patterns of HvBM5A correlate with meristem differentiation and agree with previous
reports in the literature (Yan et al. 2003; Danyluk et al., 2003).
Finally, the
vernalization requirement phenotype data from the cross of Morex x Hordeum vulgare
subsp. spontaneum show an inheritance pattern in agreement with a two-locus,
epistatic model. Definitive proof that HvBM5A is Vrn-H1 will require forward and/or
reverse genetics strategies, although using a MADS-box that affects plant growth and
development might be a challenge.
58
Differences in vernalization requirement, f due to HvBM5A, are regulatory and not
structural
Differences in vernalization requirement are due to regulatory differences in
HvBM5A. The coding regions of all ten genotypes surveyed are identical. HvBM5A
reveals a high degree of Vrnl gene sequence conservation, and presumably function,
in the Triticeae. Searching for promoter differences in the ten representative barley
genotypes, we found that the CArG-like motif, reported to be an important regulatory
site (Yan et al., 2003), to be invariant in all ten accessions. Differences correlated with
growth habit were observed, as well as genotype-specific differences. No obvious
motifs were identified that represented promising candidates for the role of regulating
HvBM5A by ZCCT-H. We genotyped the array for an intron 1 large deletion in all ten
barley varieties and found that all the winter and facultative genotypes, and OSU1 1
(the Hordeum vulgare subsp. spontaneum accession), contain the full HvBM5A intron
1 version relative to the two deleted spring genotypes. Since winter habit is the
ancestral form in the Triticeae, the intron
1
deletion may be of functional and
evolutionary significance. We have further reduced this critical region of this deletion
to 2.8 kb region, by sequencing and aligning representative genotypes with a diploid
and a hexaploid wheat accession. The first 436-bp region showed the highest sequence
conservation, suggesting that it may be the site of conserved regulatory elements
possible for growth habit. Biochemical and/or transgenic experiments will be
necessary to establish the role of these motifs in the regulation of HvBM5A. The main
question remains: "is Vrn2 the trans-acting regulatory factor of Vrnl or is Vrn2 a
cofactor in a complex that effects the DNA binding of Vrnl?"
59
The Vrn-H2 deletion explains spring andfacultative growth habit
Spring and winter growth habit is defined based on the presence or absence of
a vernalization requirement, and deletion of Vrn-H2 in spring barley accessions (Yan
et al. 2004) provides a genetic mechanism to explain the phenotype. A complete
association of the deletion of the tightly linked ZCCT-H gene family (one of which,
ZCCT-H1 is the most likely candidate for Vrn-H2) with spring growth habit was
observed. The ZCCT-H genes were also deleted in Dicktoo and 88Ab536 (facultative
genotypes) and Tremois (a genotype with conflicting growth habit descriptors). We
propose that the term facultative growth habit is descriptive of genotypes that are low
temperature-tolerant and have "winter" alleles at Vrn-H1, but lack the repressor
encoded by Vrn-H2. This could lead to re-classification of Tremois as a facultative.
Tremois meets the Vrn allele architecture criteria and we are currently characterizing
its low temperature tolerance and other winter hardiness related phenotypes in
controlled environment tests.
The Vrn2/Vrnl two-locus model provides a genetic explanation for vernalization
requirement QTL
Vernalization requirement QTL are reported from a number of barley mapping
populations, and Vrn loci were hypothesized to be candidate genes for these QTL
effects (Hayes et al., 1997). Our characterization of the Vrn-H1 and Vrn-H2 locus
genotypes in mapping population parents explains QTL effects in all reported mapping
populations. The finding that our Vrn-H1 and Vrn-H2 allele data are sufficient to
explain all reported vernalization requirement QTL data are additional evidence that
Vrn-H3 may be a determinant of vernalization response only in a limited range of
barley gerrnplasm, as reported by Takahashi and Yasuda (1971). We provide
preliminary data that a second form of HvBM5 (HvBM5B) is a candidate for Vrn-H3,
although additional experiments are clearly necessary. The pseudogene structure of
HvBM5B, and the lack of expression of this gene in Strider, Dicktoo, and Morex
support the QTL data.
Vrn loci and other winter hardiness phenotypes
The localization of Vrn-H1 in the genome is coincident with QTL for the
winter hardiness-related traits on chromosome 5H (Hayes et al., 2003).
In the
Triticum spp. low temperature tolerance effects coincide with Vrnl (Crosatti, et al.,
2003). It still needs to be determined if these are separate loci or if they are the
pleiotropic effects of the Vrn-H1 locus.
Relevance offindings
The barley germplasm pool, including wild progenitors, contain abundant genetic
variation for the components of winter hardiness (Karsai et al., 2004). Since barley is a
simple genetic system, tools have been developed for genetic and molecular analysis
(reviewed in Hayes et aL, 2003), including multiple mapping populations, arrayed
BAC clones (Yu et al., 2000), a large EST database, and a microarray chip (Close et
al., 2004). These tools should be useful for the genetic dissection of vernalization. For
example molecular markers have been used to introgress novel alleles from Hordeum
61
vulgare subsp. spontaneum into the elite cultivated germplasm (Matus et al. 2003).
The judicious application of the aforementioned genomic tools should allow for the
rapid development of gene-specific markers to introgress new allelic combinations
from wild accessions. This will increase the number of parental allele combinations in
elite lines during conventional and marker assisted breeding (Francki and Appels,
2002), and thus contribute to agricultural diversity.
A breeding objective of some programs in temperate climates, in creating fallsown barley or wheat, is to breed varieties that do not require vernalization, but that
are cold tolerant. A lack of vernalization requirement facilitates accelerated
gerrnplasm advance. Photoperiod sensitivity in such genotypes would be desirable in
order to prolong the vegetative period sufficiently to reduce risk of early spring low
temperature injury. Therefore, the identification of the genes involved in vernalization,
and other winter hardiness related traits, will be very useful to breeders as genespecific markers for marker assisted breeding.
62
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Appendix
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70
(continued)
HVBM5A-Kk
HVBK5A-Nu
HvBK5A-St
HvPM5A-Ig
HvBM5A-Dt
HvBI'f5A-Ab
HvPM5A-Tr
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HvBM5A-Kk
HVBM5A-Nu
HvBMSA-St
HVBM5A-Ig
HvflM5A-Dt
HVBM5A-Ab
HvBM5A-Tr
HVBM5A-01
HVBM5A-Mx
HVBIf5A-Ha
Figure Al.
71
(Continued)
Figure Al. Alignment of the HvBM5A coding sequence in ten barley varieties with
different growth habit. The ten barley varieties are represented as follows: winter types
Kompolti korai (Kk), Nure (Nu), Strider (St), and Igri (Ig); facultative types Dicktoo
(Dt) and 88Ab536 (Ab); spring types Morex (Mx) and Harrington (Ha); Nure (Nu)
and the Hordeum spontaneum OSU1 1 (011)
77
(Continued)
Figure A2. Alignment of the HvBMSA promoter sequence in ten barley varieties
differing in growth habit. Motifs that might be of importance and are conserved in all
barley accessions are siø, a C-repeatldehydration responsive element (CRT/DRE)
found in the promoter of cold regulated (çOR) genes and known to be target of Crepeat binding factor (CBF) genes,
CRI/DRE regulatory element targeted by
the barley HvCBF2 gene,
CArG consensus sequence found in the
promoter of Arabidopsis SOC 1 which is the MAD S-box flowering-time gene, where
FLC is a flowering repressor and a component of the vernalization pathway binds, and
binds directly to this type of GarG site and blocks transcriptional activation of SOC1
an alternate CArG
by CONSTANS (CO) (Hepworth et al., 2002),
consensus sequence. The motifs that are not conserved and might be interru ting a
regulatory element are
a
an ABA responsive element,
A
A
£
A
Putative
Methyl Jasmonate responsive element. Putative TATA-box:
transcription start: ; Start codon:
. The ten barley varieties are represented as
follows: winter types Kompolti korai (Kk), Nure (Nu), Strider (St), and Igri (Ig);
facultative types Dicktoo (Dt) and 88Ab536 (Ab); spring types Morex (Mx) and
Harrington (Ha); Nure (Nu) and the Hordeum spontaneum OSU1 1 (011).