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Vernalization Gene Architecture as a Predictor of Growth Habit in Barley
By
Douglas Heckart
A thesis submitted
to
Oregon State University
In partial fulfillment of
the requirements for the degree of
Bachelors of Science
in
Bioresource Research
Applied Genetics
and
Crop and Soil Science
Crop Breeding and Genetics
Presented
June 19, 2006
©Copyright by Douglas L Heckart
June 19, 2006
All rights reserved
Bachelor of Science thesis of Douglas Heckart presented on June 19, 2006
Approved:
Dr. Patrick Hayes
Primary Faculty Mentor
Date
Dr. Thomas Chastain
Secondary Faculty Mentor
Date
Ann Corey
Research Advisor
Date
Dr. Anita Azarenko
Director of Bioresource Research Program
Date
I understand that my thesis will become part of the permanent collection of Oregon Sate
University Libraries. My signature below authorizes release of my thesis to any reader
upon request.
Douglas L. Heckart
Date
An Abstract of the Thesis of
Douglas L Heckart for the degree of
Bachelor of Science in
Bioresource Resource Research and Crop and Soil Science
presented on June 19, 2006.
Title: Vernalization Gene Architecture as a Predictor of Growth Habit in Barley
Abstract approved: _Dr. PatrickHayes___________________________________
Primary Research Mentor
Date
Winter hardiness in barley (Hordeum vulgare) is a trait targeted by breeding
programs in order to expand the potential area of adaptation of fall-sown cereals.
Vernalization requirement is an important factor in winter hardiness. A vernalization
requirement is an extended period of low temperature required for transition from the
vegetative to reproductive states. A model involving two vernalization genes that interact
in an epistatic fashion to determine the vernalization phenotype has been proposed. To
test this model, the vernalization phenotype was compared to the vernalization allele
genotype for each entry in a breeding program nursery grown under fall- and spring-sown
conditions. The number of growing degree days (GDD) from planting to Feekes growth
stage 10.5 was recorded for each line in both experiments. Lines that did not reach
Feekes 10.5 when spring-sown were assigned values of 1000 GDD. To calculate the
growing degree day vernalization coefficient (GDD-VC), the fall sown GDD value was
subtracted from the spring sown value for each line. The GDD-VC values ranged from
139 to 812. Facultative phenotypes were classified as <309 GDD-VC or less and winter
phenotypes >409 and above. Genotypes were then assessed according to their
vernalization allele configuration at the VRN-H1 and VRN-H2 loci. All lines had the
“winter” allele at HvBM5A, the candidate gene for the VRN-H1 locus. Out of 54 lines, 46
had the “winter” allele at VRN-H2, where the ZCCT-H gene cluster is the candidate,
classifying them as “winter” genotypes. Winter and facultative phenotypes were
compared to winter and facultative genotypes. The phenotype was accurately predicted
from genotype 94% of the time. Possible reasons for the <100% accuracy include
residual heterogeneity and difficulty in classifying the growth habit of some lines under
spring-sown conditions.
Table of Contents
Page
Introduction
2
Methods and Materials
10
Results
14
Conclusions
19
Tables and figures
21
Literature cited
28
Acknowledgements
31
1
Introduction
Barley (Hordeum vulgare subsp. vulgare) is a crop species of worldwide
importance and an important genetic model for the Triticeae. Barley was one of the first
domesticated crops originating from the Fertile Crescent approximately 10,000 years ago
(Badr et al.,, 2000). This crop is important throughout the world because it has numerous
end uses. In South America and parts of Asia, barley is traditionally used as a food crop.
In the United States and Europe, barley is predominately used as an animal feed, although
the most valuable use is as the primary source of malt for beer (Hayes, 2003). The
inflorescence of barley has either one or three fertile florets per rachis node. The former
leads to a “two-row” inflorescence and the latter to a “six-row” inflorescence”. These
two inflorescence types define the major germplasm groups of barley.
The division into the two-row and six-row germplasm groups is supported by
numerous genetic diversity studies e.g. Matus et al., (2002). These genetic diversity
studies were possible due to the abundant genetic resources available in barley. Barley is
diploid (2n = 14), making it a model system for more genetically complex members of
the Triticeae, such as hexaploid wheat. The genetic resources in barley include 48
comprehensive linkage maps (GrainGenes, wheat.pw.usda.gov/gg2/index.shtml), mapped
QTLs, 348,620 Expressed Sequence Tags (www.harvest.ucr.edu) and 46,764 cloned
mRNA (from “barleygene” NCBI). These genetic resources facilitate analyses of a range
of traits, including the genes that define the major growth habit forms of barley.
The growth habit forms of barley are spring, facultative, and winter.
Agronomically speaking, spring forms are planted in the spring and harvested late
summer; winter forms are planted in the fall and harvested the following summer; and
2
facultative forms can be planted in the fall or spring. Growth habit is important because it
determines the range of adaptation. Winter and fall-sown facultative varieties generally
yield more than spring-planted forms, provided that they have sufficient winter hardiness.
However, a lack of winter hardiness limits the acreage of fall-sown cereals (Fowler et al.,
2001). The ancestral growth habit of barley is the winter habit. The spring growth habit
developed through loss of function of vernalization genes (Yan et al., 2004).
In order to expand the potential area of adaptation of fall-sown cereals, winter
hardiness has been the subject of intensive research in the Triticeae (Skinner et al., 2005).
The three components of winter hardiness are low temperature tolerance, vernalization
requirement, and photoperiod sensitivity (Skinner et al., 2005). The genetics of low
temperature tolerance and photoperiod sensitivity were reviewed by Skinner et al., (2005)
and Szucs et al., (2006). The focus of this research is on vernalization requirement.
A vernalization requirement is an extended period of low temperature required for
transition from the vegetative to the reproductive state. Without vernalization, genotypes
that have a requirement will eventually flower, but the flowering time will be delayed by
months and the flowering will be erratic (Karsai et al., 2001). The vernalization
requirement ensures plants will be in a vegetative state during the winter, and maximum
cold tolerance is achieved when plants are in a vegetative condition (Fowler et. al., 2001).
An explicit relationship between growth habit and vernalization requirement was
recently proposed by Von Zitzewitz et al., (2005) based on the cloning of candidate genes
for the VRN-H1 and Vr-H2 loci in a diverse sample of barley germplasm. According to
this model, winter forms require vernalization due to having the “winter” form of the
allele at the VRN-H1 loci and dominant “winter” allele at the VRN-H2 loci. “Winter”
3
alleles refer to DNA sequences encoding a competent repressor of VRN-H1 at the VRNH2 locus and a competent binding site for the repressor at the VRN-H1 locus. Spring
habit genotypes do not require vernalization due to a complete deletion of the gene
encoding the repressor and/or a deletion of the repressor binding site in VRN-H1.
Facultative genotypes lack the DNA sequence encoding the repressor but have a
competent repressor binding site in VRN-H1. Facultative genotypes are often sensitive to
short photoperiod, which accomplishes the same effect as vernalization: it maintains
plants in a vegetative state during the winter (Mahfoozi et. al., 2001). This
comprehensive model for the genetics of vernalization requirement is built on nearly
forty years of prior research.
Takahashi and Yasuda (1970) proposed a three locus model to explain the genetic
basis of spring and winter growth habit. The loci were named “Spring habit, (Sh) and
were assigned linkage map positions on chromosomes 4 (Sh1), 5 (Sh3), and 7 (Sh2).
Variation in Sh3 (now Vrn3), is observed only in rare barley germplasm from extremely
high or low altitudes (Yasuda et al., 1993). Barley chromosome numbering nomenclature
has changed such that 4 = 4H; 5 = 1H; and 7 = 5H. Furthermore, the loci are now
designated as “Vernalization, Vrn” and the locus numbering has also changed. The
renumbering is based on the cloning of candidate genes in Triticum monococcum (Yan et
al., 2003) and barley (Dubcovsky et al., 2004; Von Zitzewitz et al.,, 2005). The current
nomenclature and chromosome assignments for barley are: VRN-H1 (5H), VRN-H2 (4H)
and Vrn-H3 (1H). Vrn-H3 has not yet been cloned, but since allelic variation is not seen
in most germplasm, the genetic control of vernalization is thought to be best explained by
a two-locus model in most cases.
4
The genetic basis of the two-locus epistatic interaction in barley was described in
detail by Von Zitzewitz et al., (2005). The candidate genes of interest are the ZCCT-H
gene family at the VRN-H2 locus and the HvBM5A gene at the VRN-H1 locus. The
ZCCT-H gene family consists of three physically linked genes (Yan et. al., 2004). The
VRN-H2 complex locus encodes a flowering repressor. The HvBM5A gene encodes a
meristem identity gene. In the first intron of this gene, there is a binding site for the
repressor encoded by VRN-H2. To recapitulate the relationship of growth habit and the
genetics of vernalization, winter types require vernalization because they have the VRNH2 gene that encodes the repressor and they have Vrn-H1 alleles that have a binding site
for the repressor. Facultative genotypes do not require vernalization because even though
they have VRN-H1 alleles with the repressor binding site, they are completely lacking the
VRN-H2 gene that encodes the repressor. Spring types do not require vernalization
because they have VRN-H1 alleles that do not have a repressor binding site; the VRN-H2
gene may or may not be present.
These genetic models for vernalization requirement require accurate measurement
of the vernalization requirement phenotype. The vernalization requirement phenotype
can be calculated from data collected on plant material grown under controlled or field
environments. In either case, the basic measure of vernalization requirement is the
difference between the growth and/or development of plants with and without
vernalization. Controlled environments have been used for measuring vernalization
requirement in oat (Holland et al.,, 2002) and barley (Karsai et al., 2005). In the latter, the
vernalization requirement was calculated as the difference, in number of days until
flowering, between unvernalized and vernalized plants grown under the same light
5
intensity, photoperiod duration, and temperature. The principal drawbacks to controlled
environments are high cost and the limited number of plants that can be assayed. An
advantage, and a drawback, to controlled environments is that they cannot reflect the
complex environmental cues plants receive under field conditions. Under field
conditions, vernalization requirement can be measured as the difference in the number of
days to flowering between spring-sown and fall-sown experiments. As described by
Francia et al., (2003), the data from the two sowing dates can be standardized by using
the number of days from April 1 to flowering (heading) rather than the number of days
from planting. Genotypes that do not flower under spring sown conditions are assigned a
high value of 1000 days. A problem with using calendar days to heading is that it does
not take into account the role of temperature and its effects on plant growth and
development. A solution is to use the difference in the number of growing degree days
(GDD) between the spring and fall plantings (T. Chastain, personal communication,
2006). Regardless of whether the plant material is grown under controlled environment or
field conditions, and whether or not GDD or calendar days are used, there are “maturity
per se” genes that can influence the time elapsed between emergence and flowering
(Laurie, 1997)
An extreme example of the effect of such maturity genes is that of rice
transformed with the maturity gene LEAFY from Arabidopsis. Transgenic plants were 30
days earlier to flower than the wild type (He et al.,, 2000). Another complication is that
plants may flower without vernalization but the flowering pattern may be so erratic that it
would be agronomically unacceptable to consider such plants as “spring” or “facultative”.
An alternative measure of vernalization requirement that should avoid the confounding
6
effects of maturity per se genes and erratic flowering is the final leaf number (FLN).
Fowler et al., (2001) have used this measure successfully with plants grown with
hydroponics under controlled environment conditions. This measure of the vernalization
phenotype, however, would be costly to implement with field grown material., Finally,
spring-sown genotypes can simply be assigned a rating based on an integrative,
subjective scale of growth habit at a given time point. The advantage of such an approach
is that it is rapid and may be suitable for agronomic classification, if not for genetic
analysis. In the Oregon State University Barley Breeding Program, spring-sown plots
from the winter and facultative breeding program are rated on a 1 – 9 scale, where 1 =
maturity comparable to the spring habit check variety, 5 = normal growth and
development but late maturity, and 9 = only vegetative growth.
Understanding the genetics of vernalization requires scrupulous phenotyping, as
described in the preceding section, and it also requires cost-effective and reliable
genotyping. There is a range of techniques available for determining differences in DNA
sequence between varieties. While complete sequencing of the gene in each variety is
possible, it is too expensive and may be unnecessary. For example, the candidate gene
for VRN-H1, HvBM5A, is 11kb (spring), 17kb (winter) and the key region that
differentiates winter and spring alleles is 435bp (Von Zitzewitz et al., 2005). In the case
of the candidate for VRN-H2, the genes of interest (ZCCT-Ha and ZCCT-Hb) form the
tandemly linked ZCCT gene family of 1.7kb each. The spring allele is null: the gene
family is completed deleted (Dubcovsky et al., 2005). Therefore, the Polymerase Chain
Reaction (PCR) can be used with gene-specific primers for the critical region of HvBM5A
and to determine the presence or absence of the ZCCT gene family (Von Zitzewitz,
7
2005). Primers for the HvBM5A gene were designed to amplify the repressor binding site
that distinguished winter and facultative (presence0 from spring (absence) genotypes. In
the case of the VRN-H2 locus, the gene-specific primers function also as a dominant
marker since an amplicon is obtained only when the ZCCT gene family is present.
Dominant markers are problematic because the absence of an amplicon may be due to
absence of the gene, or to failure of the PCR. Therefore, in the case of dominant
markers, the assays may need to be repeated in order to have complete confidence in the
case of the null allele.
The two-locus epistatic genetic model proposed by Von Zitzewitz et al., (2005)
was based on comparison of gene sequences between genotypes whose vernalization
requirement had been previously determined in controlled environment and field tests.
Although the model is compelling, it remains to be validated. There are three approaches
to validation, each with advantages and disadvantages. One approach uses transgenic
technology to either constitutively or transiently over-express and under-express target
genes (Loukoianov et al., 2005). This approach requires considerable time and expense,
since only a limited number of barley genotypes are amenable to transformation. An
alternative genetic approach that has the advantage of assaying candidate genes in a
native genomic context is monitoring phenotype and genotype in progeny segregating for
alleles at one or both Vrn loci. Szucs et al., (personal communication) have taken this
approach and determined Vrn allele type and vernalization requirement in three F2
populations derived from crosses of contrasting parental genotypes. The third approach,
and that taken with this project, is to determine Vrn allele type and vernalization
requirement in an array of breeding program germplasm derived from crosses of parents
8
known to differ in Vrn allele type and growth habit. In this project 46 breeding lines in
the Oregon Elite Line Trial (ORELT) were derived from crosses involving four parents.
Strider and Kold are winter varieties (VRN-H1 recessive,VRN-H2 dominant); 88Ab536 is
facultative (VRN-H1 recessive,VRN-H2 recessive); and Orca is a spring type (VRN-H1
dominant, VRN-H2 recessive). In this project, these lines were genotyped for Vrn alleles
and phenotyped for Vrn requirement in order to answer the question “can the two-locus
Vrn genetic model be used to predict the growth habit of experimental lines in a breeding
program?” In addition, two previously uncharacterized varieties from the Pacific
Northwest – Hundred (Washington) and Eight-twelve (Idaho) – were included in the
genotyping and phenotyping assays.
9
Methods and Materials
Germplasm
A total of 427 lines in the Oregon State University Barley Breeding Project, were
phenotyped for this research. Subsequently, this research focused on one breeding
nursery – the Oregon Elite Line Trial (ORELT). The varieties and experimental lines in
this nursery, and their pedigrees, are shown in Table 1. There were 54 entries in the trial;
Strider was repeated twice, so there are 53 unique entries. There were six varieties
(88Ab536, Kold, Strider, Eight twelve, and Maja) and 47 experimental lines.
The single replicate ORELT nursery was planted in the fall and in the spring at
the Hyslop Agronomy Farm, near Corvallis, Oregon. The soil type at this location is
Woodburn silt loam with a 0 – 3% slope. The fall-sown nursery was planted on October
14, 2004. Each entry was represented by one 4.6 meter long, six-row plot. The spring
sown-nursery was planted on March 9, 2005. Each entry was represented by one 1.6
meter long, one-row plot. Fertilization and weed control were in accordance with
recommended practice for the location. Supplemental irrigation was applied only to the
spring-sown nursery.
Phenotyping
The growth stage was recorded on 211 fall-sown plots and 216 spring sown plots.
However, as the study focused on the ORELT, the subsequent information is specific to
the 54 entries that were the basis of this research.
Growth staging: The Feekes growth stage for each entry was recorded every two weeks
(Large, 1954). No further measurements were taken after Feekes 10.5, when the
inflorescence was fully emerged.
10
Growing degree day vernalization coefficient: The GDD values used in this experiment
were obtained in from the Oregon State University Hyslop Agronomy farm website
(http://www.ocs.orst.edu/corv_dly.html). The GDD values were calculated each day. The
high and low temperatures were averaged and the base temperature was subtracted to
produce the daily GDD value. The base temperature used in this calculation was 10°C.
The number of GDD from planting to Feekes 10.5 was calculated for each entry in the
fall and spring-sown experiments. Growing degree days Entries that failed to reach
Feekes 10.5 in the spring-sown nursery were assigned a GDD value of 1000. The
vernalization requirement was calculated as the difference in these GDD values between
the spring and fall sown experiments. This is referred to as the Growing Degree Day
Vernalization Coefficient (GDD-VC). An example of this calculation is shown in Table
2. The spring-sown ORELT was also rated for “growth habit” when the spring variety
“Baronesse” was at Feekes 10.5. A 1 – 9 scale was used to characterize growth habit,
where 1 = spring growth habit and maturity phenotype comparable to “Baronesse” and 9
= a completely vegetative growth habit and maturity phenotype comparable to the winter
check “Strider”.
Genotyping
Genotyping was performed at two locations: the USDA/ARS regional genotyping
lab at Pullman, Washington and the Oregon State University barley genotyping facility.
DNA was extracted from independent seed samples at each location. DNA was extracted
from a single plant using a QIAGEN DNeasy miniprep kit (Qiagen, Valencia, CA) at
OSU. These genomic DNA samples were used for assay of the candidate genes for the
VRN-H1 and VRN-H2 loci.
11
At Oregon State University, primers were designed to test for the presence or
absence of ZCCT-Ha and ZCCT-Hb. These two genes are very similar and the PCR
amplifies a 307bp fragment for ZCCT-Ha and a 273bp fragment for ZCCT-Hb. The
forward primer sequence was 5’ CCT AGT TAA AAC ATA TAT CCA TAG AGC 3’
and the reverse primer sequence was 5’ GAT CGT TGC GTT GCT AAT AGT G 3’. The
winter allele has an expected size of 307bp + 273bp and the spring allele an expected size
of 0bp. For VRN-H1, primers were designed to amplify the intron 1 region of HvBM5A
that is hypothesized to be the binding site for the repressor encoded by VrnH-2 (Von
Zitzewitz et al.,, 2005). The forward primer sequence was 5’ TGA GGG TAT GAG
TGG CGC TAG 3’and the reverse primer sequence was 5’ TCT CAT AGG TTC TAG
ACA AAG CAT AG 3’. The winter allele has an expected size of 435bp and the spring
allele an expected size of 0bp. The PCR amplicons were separated on 1% agarose and
stained with ethidium bromide. At the USDA/ARS lab and OSU barley lab, Corvallis
OR, the presence or absence of the ZCCT and HvBM5A genes in each ORELT line was
evaluated. The assays performed at the two laboratories were considered to be
independent replicates. In the case of the dominant alleles at HvBM5A, the allele scoring
between the two labs was compared directly. In the case of the ZCCTa and ZCCTb
dominant markers, results from the two labs were compared and the presence of an
amplicon at either of the two labs was considered to be diagnostic of the “winter” allele.
The ZCCT-H genotyping assay was repeated again at Corvallis and produced the same
allele configuration.
The predictive power of the genotyping was assessed by assigning a growth habit
classification (winter, facultative, or spring) to each genotype based on the nomenclature
12
of Von Zitzewitz et al., (2005). The growth habit assignments were then compared with
the vernalization requirement phenotype, as measured by GDD. The number of correct
predictions was divided by the total to determine the percentage of accurate predictions.
Data were recorded in Excel spreadsheets and this software was used for all calculations
and construction of phenotypic frequency distributions.
13
RESULTS
Growing conditions in both the fall-sown and spring-sown field trials were
adequate for scoring the phenotypes recorded in this study. No significant winter injury
was observed in the fall-sown trials. High winds and heavy spring rains led to significant
lodging in the fall-sown trials, but these conditions occurred after Feekes 10.5 and
therefore, did not affect the estimation of growth stages. The number of GDD from
planting to Feekes 10.5 in fall and spring-sown trials, and calculated GDD vernalization
coefficients (GDD-VC) for the check varieties and the experimental line (Kab 47),
replicated across multiple experiments are shown in Table 3. Standard errors for
88Ab536, Kab 47, and Strider were quite high because the growth stage data were taken
at two-week intervals. For instance, even if an entry was close to 10.5 at the date of
recording, it was not recorded as having reached that stage until the date of the next
reading. In the case of 88Ab536, Feekes 10.5 was reached at 122, 188, and 188 GDD in
the Oregon Elite Line Trial (ORELT), the Winter Barley Advanced Yield Trial
(WBADV), and the Winter Barley Preliminary Yield Trial (WBPYT), respectively.
When the Feekes growth stages were recorded for entries in these three nurseries on April
30, 2005, 88Ab536 in the ORELT nursery had attained Feekes 10.5, whereas in the
WBADV and WBPYT nurseries it was staged as 10.4. Only a few dyas rather than two
weeks were needed for 88Ab536 to attain Feekes 10.5 in WBADV and WBPYT.
By way of comparison, Ms. Ann Corey, Senior Research Assistant with the OSU Barley
Project, took daily notes on heading date in the same experiments and recorded a Julian
heading date of 99 days for 88Ab536 in each of the three experiments. For consistency,
the fall-sown GDD value for the ORELT was used for calculating the GDD-VC for each
14
of the check varieties and Kab 47, rather than the mean value. This value is shown in
parentheses in Table 3.
Under fall-sown conditions, there was a large range in the number of GDD from
planting to Feekes 10.5. There was a difference of 606 GDD between the earliest
(88Ab536 and Kab 47) and latest (Hundred and Kold) varieties. All check varieties
except Kold, Strider, and Hundred reached Feekes 10.5 under spring-sown conditions.
According to the spring-sown GDD data, Kold, Strider, and Hundred are the three
varieties that would be considered to have a vernalization requirement because they did
not flower within an agronomically acceptable time frame. These varieties had the largest
values GDD-VCs (812, 715, and 715 respectively). By way of comparison, the spring
habit variety Orca had a GDD-VC of 206. The other check varieties, and Kab 47, had
GDD-VCs between those of Orca and Kold. Thirty-five experimental lines did not reach
Feekes 10.5 in the spring-sown experiment (Figure 1). These entries with GDD-VCs
greater or equal to those of Strider, Kold, and Hundred can be considered to have a
vernalization requirement. Five entries, including the variety Eight twelve, had
intermediate GDD-VCs of 409 and would be too late maturing for agronomic
performance under spring-sown conditions and are therefore also considered to also have
a vernalization requirement. Genotypes with GDD-VCs between 139 and 302 are
considered facultative.
The results from the amplification of the VRN-H1 critical region with allelespecific primers were in complete agreement between the USDA/ARS and OSU labs. All
genotypes showed the expected 435bp product, which is the “winter” type allele
according to Von Zitzewitz et al., (2005). The fact that the previously uncharacterized
15
winter varieties Hundred and Eight twelve have the winter allele at this locus is further
evidence for the role of the VRN-H1 critical region as a binding site for the repressor
encoded by VRN-H2 (Von Zitzewitz et al.,, 2005). All experimental lines in the ORELT
are descended from crosses among Strider, 88Ab536, Kold, and Orca (see pedigrees in
Table 1). Of these parents, all have been genotyped, except for Orca, and all have the
winter allele at VRN-H1. The VRN-H1 allele architecture of Orca can be inferred based
on parentage and phenotype. The parents of Orca are Calicuchima and Bowman.
Calicuchima, a parent of Orca, does not have a vernalization requirement and was
genotyped and identified to have the VRN-H2 gene and a unique “spring” allele at VRNH1 (P. Szucs, personal communication). Bowman is a spring variety from North Dakota
and it is unlikely that it has a winter allele at VRN-H1. Therefore, it is reasonable to
hypothesize that Orca has a spring allele at VRN-H1.
Fourteen ORELT entries have Orca in their pedigree, yet all of these lines have
the Strider (winter) allele at VRN-H1. This is probable evidence of stringent selection
against the inferred Orca allele at this locus. The Strider/Orca lines were developed by J.
Von Zitzewitz for his Bio Resource Research (BRR) undergraduate thesis at Oregon
State University. His goal was to combine winter hardiness (from Strider) with Barley
Yellow Dwarf Virus (BYDV) resistance from Orca. Only the lines surviving a low
temperature event at Corvallis in December 1998 were advanced, and it is likely that
winter survival was associated with the Strider allele at VRN-H1. According to Skinner
et al., (2005), the VRN-H1 locus is associated with low temperature tolerance and is a
candidate for the low temperature tolerance QTL Fr-H1. The linked cluster of HvCBF
loci is a candidate for the Fr-H2 QTL. Thus, selection for winter survival in the
16
segregating progeny of Strider/ Orca crosses would have led to fixation of the Strider
alleles at loci in this region of the genome.
There was not complete agreement between labs for the VRN-H2 locus. At the
USDA/ARS lab, no amplification was obtained for ORELT lines 17, 23, 29, 41, 48, 52,
53 and at OSU no amplification was obtained for ORELT lines 26 and 27. Possible
explanations for these discrepancies between laboratories are: (i) PCR failure, (ii) sample
contamination, and (iii) heterogeneous samples. PCR failure is a problem with dominant
markers, such as the primers for VRN-H2, since the ZCCT gene family is either present or
completely deleted. Sample contamination is always a possibility, but in each laboratory
standard operating procedures are designed to minimize the likelihood of this happening.
Entries in the ORELT were at the F6, or more advanced generation at the time of DNA
extraction. However, some within-line heterogeneity is always possible. Unless winter
breeding lines are routinely screened under spring-planted conditions, it is possible that
“winter phenotype” lines are actually mixtures of winter and facultative types. DNA
extractions are performed on a single plant from a single seed representing each variety
or selection. In the case of heterogeneous varieties or lines, the random selection of the
seed for DNA extraction could lead to contrasting conclusions regarding the presence or
absence of VRN-H2. The characterization of growth habit based on spring-sown
phenotype would depend on the percentage of seeds within the seed lot positive for VRNH2. PCR failure and/or heterogeneous seed lots are the most likely explanations for the
discrepancies, since the VRN-H2 assay involves a dominant marker. For the purposes of
this project, the presence of a PCR product at one or both labs was considered to be
diagnostic of the presence of VRN-H2. By this criterion, the OSU data would be correct
17
for the seven lines that failed to amplify at the USDA/ARS lab and the USDA/ARS data
would be correct for the two lines that failed to amplify at the OSU lab. This hypothesis
was tested by performing new, independent, PCR reactions at OSU. All lines that
previously amplified at OSU, but not at USDA/ARS, were positive for VRN-H2 and the
two lines that were negative at OSU, remained negative for this allele. These genotyping
results were then considered in the context of the phentoyping results.
Since all ORELT entries have the winter allele at VRN-H1, the genetic model to
account for vernalization requirement reduces to allelic variation at the VRN-H2 locus
and all entries in the experiment can be classified as winter or facultative, per the
definition of Von Zitzewitz et al., (2006). This growth habit classification, where “w” =
winter and “f” = facultative is shown for each entry in the ORELT, together with the fall
and spring-sown GDD values, and the GDD-VC in Table 4. With the exception of three
entries – ORELT 18, 23, and 24 - all genotypes with GDD-VCs > 400 are classified as
winter and those with GDD-VCs < 400 are classified as facultative. Therefore, the VRNH2 allele type is an excellent predictor of growth habit, but it failed in 3/54 cases.
ORELT 18, 23, and 24 were lines with GDD-VCs of 236, 236, and 139 respectively:
based on phenotype they are clearly facultative. However, 18 and 24 were scored as
VRN-H2 positive at the USDA/ARS lab and 24 was scored as VRN-H2 positive at the
OSU lab.
18
Conclusions
The model proposed by Von Zitzewitz et al., (2005) has essentially been validated
by this study. The GDD-VC phenotype was predicted by the Vrn allele phenotype in 51
out of 54 cases. This accuracy level is quite acceptable and indicates that the Vrn
phenotype is a suitable target for marker assisted selection (MAS). However, there
remains the question of why the correlation was not perfect. There are several possible
explanations for the discrepancy. These include residual heterogeneity, DNA
contamination, or ambiguity in rating the phenotype. The USDA/ARS lab has been
contacted to determine if reserve tissue is available for re-extraction of DNA. Residual
heterogeneity will be assessed in the summer of 2006, as the ORELT is planted for a
second time for assessment of vernalization requirement. Residual heterozygosity is
hypothesized to account for the two lines (26 and 27) that were winter habit according to
their GDD-VC’s and have the VRN-H2 allele according to the USA/ARS data, but not the
OSU data. Ambiguity in scoring growth stages under spring planted conditions may
account for some discrepancies. For reasons that are not understood, many winter
genotypes show erratic flowering under spring-sown conditions. The plant remains in an
overall vegetative state but one or two inflorescences are formed. Technically, the plant
reaches Feekes 10.5 when these few inflorescences are at the appropriate stage. Three
ORELT entries are VRN-H2 positive, but had GDD-VC’s of 236,236, and 139
respectively. These same entries were rated 4, 7, and 7, respectively, for growth habit
under spring-sown conditions by Dr. Pat Hayes. This is a subjective rating scale where 1
= flowering at the same time as the local spring check (Baronesse) and 9 = completely
19
vegetative. These intermediate ratings confirm that the spring growth habit was not
entirely normal and the erratic growth habit could result from the presence of VRN-H2.
There are two recommendations that can be made for future research in this and
for Vrn MAS. Genotyping can be improved by augmenting the VRN-H2 genotyping with
a codominant assay. This can be done by using a linked gene that shows complete linkage
disequilibrium with VRN-H2. The HvSnf2 gene is good candidate for this type of assay
(Von Zitzewtiz et al., 2005). This gene is distal to ZCCT and is present in all “spring”,
“winter” and “facultative” genotypes.
Phenotyping methods can also be improved. There were ambiguities between
checks in different nurseries. These differences were often misleading due to the two
week period between dates when plants were evaluated. It was possible for a line to reach
Feekes 10.4 on a measurement date and 10.5 several days later, yet the date recorded
would have been two weeks later. This problem can be resolved by evaluating plants
daily. In some instances there are lines that flower under spring-sown conditions, but not
in an agronomically acceptable fashion. In these cases the FLN could be measured and
compared to FLN of spring and winter checks.
20
Table 1. Varieties and experimental lines, and their pedigrees, in the Oregon Elite Line
nursery planted at Corvallis, Oregon in the fall of 2004 and spring of 2005.
ORELT No
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
Variety or Selection
88Ab 536
Kold
Strider
Kab 47
Hundred
Eight-Twelve
Maja
J2-5-1
StabBC 42-3-2
J1-8-17
J2-5-4
J2-12-2
Stab 113/Kab 43-1
J2-5-12
J1-13-1
Stab 113/Kab 50-22
StabBC 50-7-3
J2-6-19
StabBC 182-6-5
Stab 47/Kab 51-20
J2-15-1
StabBC 50-7-6
StabBC 50-9-1
StabBC 50-7-1
Stab 47/Kab 51-9
StabBC 50-9-2
Stab 47/Kab 51-7
StabBC 42-4-5
StabBC 182-4-2
StabBC 42-3-8
Strider
J2-17-2
StabBC 42-4-7
J1-20-2
StabBC 42-3-5
StabBC 42-3-6
StabBC 42-3-4
StabBC 42-3-9
J2-5-11
J2-5-2
StabBC 42-4-2
StabBC 182-4-1
Pedigree
Ne 76129/Morex/Morex
1285/Astrix
OR 1860164/ Steptoe
Kold/88Ab536
Strider/88Ab536
Strider/Orca
Strider/88Ab 536
Strider/Orca
Strider/Orca
Strider/Orca
Stab 113/Kab 43
Strider/Orca
Strider/Orca
Stab 113/Kab 50-22
Strider/88Ab 536
Strider/Orca
Strider/88Ab 536
Stab 47/Kab 51-20
Strider/Orca
Strider/88Ab 536
Strider/88Ab 536
Strider/88Ab 536
Stab 47/Kab 51-9
Strider/88Ab 536
Stab 47/Kab 51-7
Strider/88Ab 536
Strider/88Ab 536
Strider/88Ab 536
OR 1860164/ Steptoe
Strider/Orca
Strider/88Ab 536
Strider/Orca
Strider/88Ab 536
Strider/88Ab 536
Strider/88Ab 536
Strider/88Ab 536
Strider/Orca
Strider/Orca
Strider/88Ab 536
Strider/88Ab 536
21
43
44
45
46
47
48
49
50
51
52
53
54
StabBC 42-3-10
StabBC 42-4-8
StabBC 42-3-7
StabBC 42-4-3
J1-8-16
StabBC 42-3-3
J2-5-3
StabBC 42-3-11
S113/K50-21
StabBC 42-4-6
StabBC 50-7-5
J1-14-2
Strider/88Ab 536
Strider/88Ab 536
Strider/88Ab 536
Strider/88Ab 536
Strider/Orca
Strider/88Ab 536
Strider/Orca
Strider/88Ab 536
Stab 113/Kab 50 - 21
Strider/88Ab 536
Strider/88Ab 536
Strider/Orca
22
Table 2. An example of calculation of the growing degree day vernalization coeffiecient
(GDD-VC). Growing degree days from planting to Feekes growth scale 10.5 for fall
planting subtracted from GDD value for spring planting.
GDD to heading
Variety
Spring
- Fall =
GDD-VC
88Ab536
424
- 122 =
302
Strider
1000
-
188
=
23
812
Table 3.Growing degree day vernalization coefficients (GDD-VCs) for the check
varieties and experimental selection (Kab 47) that were replicated in fall and spring-sown
field trials at Corvallis, Oregon in the 2004-2005 crop year.
Growing
degree day
vernalization
Spring
coefficient
Variety or
Fall sown
sown
(GDD-VC)*
selection
GDD
GDD
88 AB 536 166 (122)** + 22
424
302
EightTwelve
188***
597
409
Hundred
285
1000
715
Kab 47
166 (188)+22
424
236
Kold
285
1000
715
Maja
188
424
236
Orca
113
319****
206
Strider
212 (188)+22
1000
812
* The GDD-VC was calculated as (Spring-sown GDD - Fall-sown GDD)
** ORELT nursery values are shown in parentheses for the entries that did not have
identical GDD values in all nurseries.
***If no standard error indicated, the number of GDD was the same in all experiments.
**** One observation only
24
Table 4. Growing degree day vernalization coefficients (GDD-VCs) and growth habit
classification, based on VRN-H1 and VRN-H2 allele genotypes for commercial varieties
and experimental lines in the Oregon Elite Lines Trial (ORELT) in the 2004-2005 crop
year.
ORELT
Variety or
no.
Selection
1
88Ab 536
Fall Spring GDD-VC growth habit
Pedigree
GDD GDD
classification
Ne 76129/Morex/Morex 122
424
302
F
2
Kold
1285/Astrix
285
1000
715
W
3
Strider
OR 1860164/Steptoe
188
1000
812
W
4
Kab 47
Kold/88Ab536
188
424
236
F
5
Hundred
285
1000
715
W
6
Eight-Twelve
188
597
409
W
7
Stab 113
Strider/88Ab536
188
424
236
F
8
J2-5-1
Strider/Orca
285
1000
715
W
9
StabBC 42-3-2
Strider/88Ab 536
285
1000
715
W
10
J1-8-17
Strider/Orca
285
1000
715
W
11
J2-5-4
Strider/Orca
188
1000
812
W
12
J2-12-2
Strider/Orca
188
1000
812
W
13
Stab 113/Kab 43-1
Stab 113/Kab 43
188
424
236
F
14
15
J2-5-12
J1-13-1
Strider/Orca
Strider/Orca
285
188
1000
1000
715
812
W
W
16
Stab 113/Kab 50-22 Stab 113/Kab 50-22
285
424
139
F
17
StabBC 50-7-3
Strider/88Ab 536
285
1000
715
W
18
J2-6-19
Strider/Orca
188
424
236
W
19
StabBC 182-6-5
Strider/88Ab 536
285
1000
715
W
20
Stab 47/Kab 51-20
Stab 47/Kab 51-20
188
424
236
F
21
J2-15-1
Strider/Orca
188
1000
812
W
22
StabBC 50-7-6
Strider/88Ab 536
188
1000
812
W
23
StabBC 50-9-1
Strider/88Ab 536
188
424
236
W
24
StabBC 50-7-1
Strider/88Ab 536
285
424
139
W
25
Stab 47/Kab 51-9
Stab 47/Kab 51-9
188
424
236
F
26
StabBC 50-9-2
Strider/88Ab 536
285
1000
715
W
27
Stab 47/Kab 51-7
Stab 47/Kab 51-7
188
1000
812
W
25
28
StabBC 42-4-5
Strider/88Ab 536
285
1000
715
W
29
StabBC 182-4-2
Strider/88Ab 536
285
1000
715
W
30
StabBC 42-3-8
Strider/88Ab 536
285
1000
715
W
31
Strider
OR 1860164/Steptoe
188
1000
812
W
32
J2-17-2
Strider/Orca
188
597
409
W
33
StabBC 42-4-7
Strider/88Ab 536
188
1000
812
W
34
J1-20-2
Strider/Orca
188
597
409
W
35
StabBC 42-3-5
Strider/88Ab 536
285
1000
715
W
36
StabBC 42-3-6
Strider/88Ab 536
285
1000
715
W
37
StabBC 42-3-4
Strider/88Ab 536
285
1000
715
W
38
StabBC 42-3-9
Strider/88Ab 536
285
1000
715
W
39
J2-5-11
Strider/Orca
285
1000
715
W
40
J2-5-2
Strider/Orca
285
1000
715
W
41
StabBC 42-4-2
Strider/88Ab 536
285
1000
715
W
42
StabBC 182-4-1
Strider/88Ab 536
285
1000
715
W
43
StabBC 42-3-10
Strider/88Ab 536
285
1000
715
W
44
StabBC 42-4-8
Strider/88Ab 536
285
1000
715
W
45
StabBC 42-3-7
Strider/88Ab 536
285
1000
715
W
46
StabBC 42-4-3
Strider/88Ab 536
285
1000
715
W
47
J1-8-16
Strider/Orca
188
597
409
W
48
StabBC 42-3-3
Strider/88Ab 536
285
1000
715
W
49
J2-5-3
Strider/Orca
285
1000
715
W
50
StabBC 42-3-11
Strider/88Ab 536
285
1000
715
W
51
S113/K50-21
Stab 113/Kab 50 - 21
188
424
236
F
52
StabBC 42-4-6
Strider/88Ab 536
285
1000
715
W
53
StabBC 50-7-5
Strider/88Ab 536
285
1000
715
W
54
J1-14-2
Strider/Orca
188
597
409
W
26
Figure 1. Growing degree day vernalization coefficient (GDD-VC) for the eight check
varieties and 46 experimental lines in the Oregon Elite Line Trial (ORELT) based on fall
and spring-sown field trials at Corvallis, Oregon in the 2004-2005 crop year.
35
30
Number of Lines
25
20
15
10
5
0
139
236
302
409
Vrn requirement GDD-VC
27
715
812
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30
Acknowledgements
I would like to sincerely thank Dr. Patrick Hayes, Head of the Oregon State Barley
Project. His knowledge, persistence, and patience have helped me enormously throughout
this project.
I would like to thank Dr. Peter Szucs for all the time and expertise with molecular
genetics that he put into this project. He always had time for questions and was eager to
help.
I would like to thank Ann Corey for the help and guidance with the field portion of this
project.
I would like to thank others in the OSU Barley Project including Tanya Filichkina and
Kelly Richardson for always making time to answer laboratory questions.
I would like to thank Tom Chastain for his help with this project and for being an
excellent advisor and mentor.
I would like to thank Wanda Crannell for the guidance throughout the Bioresource
Research degree program and for keeping me motivated during difficult times.
I would like to thank the Crop and Soil Science Department at OSU for making this
academic experience not only interesting and fun, but a family affair. Thank you.
31