AN ABSTRACT OF THE THESIS OF
Aihong Pan for the degree of Master of Science in Crop and Soil Science presented on
February 15, 1994.
Title: Genetic Analysis of Vernalization, Photoperiod, and Winter Hardiness in Barley
(Hordeum vulgare L.)
Abstract approved:
Redacted for Privacy
Patrick M. Hayes
Winter hardiness in cereals involves a number of complex and interacting
component characters: cold tolerance, vernalization requirement, and photoperiod
sensitivity. An understanding of the genetic basis of these component traits should allow
for more effective selection. Genome map-based analyses hold considerable promise for
dissecting complex phenotypes. A 74-point linkage map was developed from one hundred
doubled haploid lines derived from a winter x spring barley cross and used as the basis
for quantitative trait locus (QTL) analyses to determine the chromosome location of genes
controlling cold tolerance, vernalization response, and photoperiod response. Despite the
greater genome coverage provided by the current map, a previously-reported interval on
chromosome 7 remains the only region where significant QTL effects for winter survival
were detected in this population. QTLs for growth habit and heading date under 16 h and
24 h photoperiod was mapped to the same region. A QTL for heading date under these
photoperiod regime also maps to chromosome 2. Contrasting alleles at these loci interact
in an epistatic fashion. A distinct set of QTLs mapping to chromosomes 1, 2, 3, and 5
determined heading date under 8 h photoperiod. Under field conditions, all QTLs
identified under controlled environment conditions were determinants of heading date.
Patterns of differential QTL expression detected during plant development, coupled with
additive and additive x additive QTL effects, underscore the complexity of trait
expression. The presence of unique phenotypes in the mapping population suggests that
coincident QTLs for heading date and winter survival are probably due to linkage rather
than pleiotropy.
Genetic Analysis of Vernalization, Photoperiod,
and Winter Hardiness in Barley (Hordeum vulgare L.)
by
Aihong Pan
A THESIS
submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Master of Science
Completed February 15, 1994
Commencement June 1994
APPROVED:
Redacted for Privacy
Associate Professor of Crop and Soil S
nce in charge of major
Redacted for Privacy
Read of Department of Crop and Soil Science
Redacted for Privacy
Dean of Graduate
Date thesis is presented
Typed by Aihong Pan for
February 15, 1994
Aihong Pan
ACKNOWLEDGMENTS
I would like to thank Dr. Patrick M. Hayes, my major professor and advisor, for
his time, advice, fmancial support, and friendship.
Thanks to Dr. T.H.H. Chen, Dr. D.N. Moss, and Dr. F.W. Obermiller for
serving on my graduate committee and reviewing this manuscript.
Thank to the barley group ( Doris Prehn, Donna Moulrooney, Ann Corey, Marry-
jo Lundsten, Marci Woff, Sue Kolar, Adeline Ozied, Odianosen Iyamabo, Ergun
Ozdemir, and Abdoulage Traore ) for all their help.
I want to thank my parents and brothers for their support and encouragement.
Finally, a special thanks to my husband, Fuqiang Chen, for his support, patience,
and understanding.
TABLE OF CONTENTS
INTRODUCTION
1
MATERIALS AND METHODS
5
RESULTS
Phenotypic trait expression
Map construction
Quantitative trait loci
QTL interaction
20
28
DISCUSSION
32
REFERENCES
38
9
9
18
LIST OF FIGURES
Figure
Page
1. Frequency distributions for heading date under three
photoperiod regimes of 100 doubled haploid lines
derived from the cross of Dicktoo x Morex.
10
2. Frequency distributions for winter survival, final leaf
number, maximum tiller number, and field growth habit
of 100 doubled haploid lines derived from the cross of
Dicktoo x Morex.
11
3. Frequency distributions for main stem height of 100
doubled haploid lines derived from the cross of Dicktoo
x Morex.
15
4. Linkage map based on 100 doubled haploid lines derived
from the cross of Dicktoo x Morex (units are percent
recombination).
16
5. Two locus QTL genotype means for heading date under 8,
16, and 24 h light.
29
LIST OF TABLES
Table
Page
1. Phenotypic correlations among heading date, growth habit
characters, and winter survival for 100 doubled haploid
lines derived from the cross of Dicktoo x Morex.
17
2. QTL peak effects for heading date of 100 doubled haploid
lines derived from the cross of Dicktoo x Morex.
22
3. QTL peak effects for growth traits, field growth habit, and
winter survival of 100 doubled haploid lines derived from
the cross of Dicktoo x Morex.
24
4. QTL peak effects of main stem height under 24 h light for
100 doubled haploid lines derived from the cross of Dicktoo
x Morex.
27
Genetic Analysis of Vernalization, Photoperiod,
and Winter Hardiness in Barley (Hordeum vulgare L.)
INTRODUCTION
Winter hardiness in cereals is the fmal expression of a number of interacting
characters that can include vernalization requirement, photoperiod response, and low
temperature tolerance. Thomashow (1990) reviewed cold tolerance physiology and
genetics research in higher plants, including cereals. Hayes et al. (1993a) recently
reported the presence of a putative multilocus cluster on barley chromosome 7 defined
by Quantitative Trait Locus (QTL) effects for a number of winter hardiness component
traits.
The genetic basis of vernalization and photoperiod response in cereals has been
the subject of extensive study for nearly 100 years. Takahashi and Yasuda (1970)
proposed that three major genes sh, Sh2, and Sh3 located on chromosomes 4, 7, and
5, respectively, were responsible for winter vs. spring growth habit in barley (Hordeum
vulgare). Multiple allelic series at each locus and complex epistatic interactions between
loci were postulated to explain the range of growth habit seen in barley germplasm
(Nilan, 1964; Takahashi and Yasuda, 1970). According to this model, the only allelic
combination giving full winter growth habit is ShShsh2sh2sh3sh3. Although this working
model is wildly accepted, none of these genes have been characterized at the molecular
level. In hexaploid wheat (Triticum aestivum), five major genes
Vml, Vrn2, Vrn3,
2
Vrn4, and Vrn5 are reported to control the vernalization response (Pugs ley, 1971, 1972;
Law, 1966). Vml , Vrn4, and Vrn3 are located on chromosomes 5A, 5B, and 5D,
respectively (Xin et al., 1988). Vrn2 is located on chromosome 2B, and Vrn5 is located
on chromosome 7B (Law, 1966). The homoeologous relationship of wheat and barley
chromosomes is such that the wheat chromosome 2 is homoeologous to barley
chromosome 2, wheat 5 to barley 7, and wheat 7 to barley 1 (Islam et al., 1981).
The most frequently employed measure of winter vs. spring growth habit in
cereals is heading date. Takahashi and Yasuda (1970) concluded that at least three
internal physiological factors vernalization response, photoperiod response and earliness
were responsible for heading date in barley. Vernalization responses are strongly
influenced by photoperiod (Roberts et al. , 1988) and photoperiod responses can be
modified by vernalization (Takahashi and Yasuda, 1970). Earliness per se has been the
subject of much research, particularly in spring habit barley. Four loci Easp, Eac, Eak,
and Ea7 located on chromosomes 3, 4, 5, and 6 respectively,
were reported to control
both maturity and photoperiod sensitivity in spring barley (Gallagher et al., 1991; Yasuda
and Hayashi, 1980; Dormling and Gustafsson, 1969; Ramage and Suneson, 1958). The
Ea locus on chromosome 2 was described by Nilan (1964) and Hayes et al. (1993b)
reported the presence of a large-effect QTL for heading date in the same chromosome
region. A lack of common markers precludes drawing definitive assignment of this QTL
effect to the Ea locus. The effects of these Ea loci in photoperiod and vernalizationsensitive genetic backgrounds are unknown.
3
Low temperature tolerance is an induced response (Thomashow,1990) and thus
hardening and maximum expression of cold tolerance are often confounded because the
two characteristics are often found in association. This has led to considerable discussion
of underlying genetic mechanisms (Cahalan and Law, 1979; Brule-Babel and Fowler,
1988). Recent evidence points to linkage rather than pleiotropy. Roberts (1989) reported
that at least two loci on chromosome 5A were involved with cold hardening in wheat.
One locus is, or is tightly linked to, the vernalization gene Vrnl, but the other locus
appears to be unrelated to the vernalization gene. In barley, Doll et al. (1989) reported
that doubled haploid lines with good winter hardiness but without a vernalization
requirement were recovered from winter x spring crosses. Hayes et al. (1993a) reported
segregation for low temperature tolerance in the progeny of a winter x spring cross,
where the winter parent was photoperiod sensitive but had no vernalization requirement.
Progress in elucidating the genetic mechanisms of winter hardiness, vernalization
and photoperiod responses is hindered by the often quantitative nature of trait expression
and complex interactions among traits. The development of molecular marker technology
and Quantitative Trait Locus (QTL) mapping offers a new approach to resolving this
complexity (Paterson et al., 1993; Lander and Botstein, 1989). By locating individual
QTLs with molecular markers, individual QTL genotypes can be determined with relative
certainty. This would allow for a dissection of complex traits, the measurement of
individual gene effects, and the measurement of gene interactions. Ultimately, higher
resolution mapping will allow for the separation of pleiotropic and linkage effects
(Paterson et al., 1990) and map-based cloning (Chang et al., 1993; Martin et al., 1993).
4
Several molecular marker maps of barley have been developed (Heun et al. , 1991;
Graner et al. , 1991; Kleinhofs et al. , 1993). These maps have been used to locate QTLs
affecting yield, quality, and disease resistance traits (Hayes et al. , 1993b; Heun et al. ,
1992; Chen et al., 1994a). Based on analyses of a winter x spring cross, Hayes et
al. (1993a) reported the presence of a QTL on the long arm of chromosome 7 that was
associated with low temperature tolerance in both field and controlled environment tests.
QTL effects for heading date under 24 h light and other winterhardiness-related traits
mapped to the same region. However, the presence of individual recombinant lines with
unique character attributes led these authors to hypothesize that the association of trait
expression was due to linkage rather than pleiotropy. Additional markers have since been
mapped in this same population, providing a greater degree of map resolution and
genome coverage. Therefore, the objectives of this work were to (i) reassess cold
tolerance QTL expression in view of greater map density and coverage, (ii) locate QTLs
for vernalization and photoperiod response, and (iii) determine the importance of epistatic
interactions among QTLs.
5
MATERIALS AND METHODS
Two barley cultivars, "Morex" and "Dicktoo", and one hundred F,- derived
doubled haploid (DH) progeny were used in these experiments. Morex, released in 1978
by the Minnesota Agricultural Experimental Station, is the U.S. six-row spring barley
malting standard. Dicktoo is a six-row winter feed barley released by the Nebraska
Agricultural Experimental Station in 1952. The pedigree of Dicktoo is unknown, and the
variety description is mixed. Under field conditions, the particular accession of Dicktoo
used in these studies displays prostrate winter growth habit and average maturity
comparable to other winter barley genotypes. The DH lines were developed by the
Hordeum bulbosum method, as described by Chen and Hayes (1989).
The 100 DH lines and the parents were phenotyped for a number of winter
hardiness component traits in field and controlled environment tests. The cold tolerance
testing procedures were described by Hayes et al. (1993a) and involved field plots at
Bozeman, Montana and Corvallis, Oregon. Growth habit ratings ( 1 = prostrate and 10
= fully erect) and heading date were recorded on a plot basis at Corvallis.
The heading dates of the 100 DH lines and the two parents were also measured
in a series of controlled environment tests involving six combinations of vernalization and
photoperiod. In all cases, the vernalization treatment consisted of six weeks of hardening
seedlings at 6°C with an 8 h light/16 h dark photoperiod regime. In all experiments,
seedlings for the unvernalized treatment were established one week prior to the end of
vernalization so that plant material from both treatments was transplanted at the same
6
time and at approximately the same growth stage. Heading date with or without
vernalization with an 8 h light/16 h dark photoperiod regime was assessed in the
phytotron facilities of the Martonvasar Research Institute (Martonvasar, Hungary) with
the light intensity and temperature program described by Tischner (1993). Heading date
with or without vernalization under 16 h light/ 8 h dark and under a continuous light
regime was determined under greenhouse conditions at Corvallis, Oregon. Ambient
temperatures were 20°±2°C, and supplemental illumination was provided by high
pressure sodium lights suspended 1.5m above the bench surface. Each genotype was
replicated twice. These six data sets will subsequently be referred to by the hours of light
duration (8h, 16h, or 24h) and a suffix indicating the vernalization treatment, where v
= vernalized and uv = unvernalized. In addition to heading date, measured as the
number of days from transplanting to awn emergence, mainstem leaf number, mainstem
height, and total tiller number were measured weekly in the 24hv and 24huv
experiments.
A total of seventy-eight markers were scored on the 100 DH progeny in a
cooperative effort involving Linkage Genetics, Inc. (Salt Lake city, Utah), Montana State
University (Bozeman, Montana), and Oregon State University (Corvallis, Oregon). The
marker nomenclature of Kleinhofs et al. (1993) is employed in this report. Briefly, the
prefixes "m ", "i" , and "ap" designate morphological, isozyme, and Sequence Tagged Site
(STS) markers, respectively. Additional detail on the STS markers can be found in
Tragoonrung et al. (1992). Restriction fragment length polymorphisms (RFLPs) were
detected with an array of anonymous cDNA and genomic clones and several known
7
function clones. The prefixes "WG", and "BCD" designate anonymous wheat genomic
and barley cDNA clones, respectively (Heun et al., 1991). The prefixes "ABC", and
"ABG" designate anonymous barley cDNA and genomic clones developed by the North
American Barley Genome Mapping Project (Kleinhofs et al., 1993). Crhl and Crh2 are
plant calreticulin loci (Chen et al., 1994b). Cr3 is a low temperature-induced cDNA
clone (Sutton and Kenefick, 1994). Dhnl, 2, 4, and 5 are Dehydrin cDNA clones
(Close,1993). The morphological markers rachilla hair (mS) and awn texture (mR) were
scored under a stereomicroscope. Isozyme and storage protein markers were analyzed as
described by Nielsen and Johanson (1986). STS markers were assayed as described by
Tragoonrung et al. (1992), and RFLP markers were assayed essentially as described by
Kleinhofs et al. (1993).
A linkage map was constructed using Mapmaker/EXP 3.0 (Lander et al., 1987;
Lincoln et al., 1992). Seventy four markers were resolved into eleven linkage groups.
Assignments of these linkage groups to the seven chromosomes of barley were made
based on previously mapped markers. QTL analyses were conducted using the interval
mapping procedures of Mapmaker/QTL 1.1 (Lincoln et al., 1992). The minimum LOD
(Loglo of the ratio between maximum likelihood estimates) threshold was usually
specified at 3.0. However, in certain cases, QTL effects significant at lower LOD scores
are reported. For the more detailed analyses of epistatic interactions, only individuals
with no crossovers between flanking markers were employed. Two tests for epistasis
were used: t-tests among populations of means and ANOVA. For the former, p < 0.05
was taken as evidence for a significant epistatic interaction. For the latter, Type HI sums
8
of squares (SAS Institute, 1988) were generated based on the two-way ANOVA of
phenotype and QTL genotype for pairs of QTLs that were individually significant in the
interval mapping analysis. A significant QTL x phenotype interaction mean square (p <
0.05) was taken as evidence for epistasis. As there is no dominance variance in a
population of completely homozygous doubled haploids, significant interactions indicate
the presence of additive x additive epistasis.
9
RESULTS
Phenotypic trait expression
The winter survival data of Hayes et al. (1993a) are presented for the sake of
reference and because of the greater genome coverage that has been achieved since the
publication of that report. Morex and approximately half of the DH lines experienced
complete mortality in Montana. The mean survival of Dicktoo was 43 % and the percent
survival of the remaining DH lines ranged from 1 to 85 (Figure 2a). The standard error
was ± 11.1 %. Less severe winter conditions at Corvallis led to survival of all DH lines,
with a range of 1 to 100 % (Figure 2b). As these estimates were based on an unreplicated
test, no standard error can be calculated. However, as demonstrated by Knapp and
Bridges (1990) the primary determinant of tests of hypotheses about QTL genotype
means is the number of replications of QTL genotypes, not the number of times each
individual is replicated. The survival of Dicktoo was 89% and that of Morex 18 % . The
growth habit profile of the population is presented in Figure 2g. Dicktoo was prostrate
and rated "3". Morex had an upright growth habit and was rated "9". There was
continuous variation in the population, ranging from "2" to "10". Three genotypes were
more prostrate ("2") than Dicktoo, and two genotypes were more upright ("10") than
Morex.
Heading date frequency distributions for the DH population in both field and
controlled environments were continuous (Figure la
1g), indicating quantitative
inheritance. Population means and ranges, and the numbers of phenotypic transgressive
(a)
(c)
Heading (8h.vernalized)
30
25
Dicktoo
20
15
15
Morex
701
80
90
1111
100
110
120
Time (days)
(b)
30
25
I. Morex
Dicktoo
20
130
10
25
rir
35
45
tit
55
5
65
i
Time (days)
(d)
Heading (8h.unvernalized)
JO
1
751
1
85
1
Heading (16h.unvernernal zed)
30
25-
Dicktoo Morex
15
10
10
Heading (24h. vernalized)
35
25
20
(e)
Heading (16h. vernalized)
30
0
.11111111411"t"
25
35
45
55
65
75
(t)
Heading (24h. unvernalized)
25
20--
Dicktoo
15-
I
10-
11111j
Morex
50
70
I
1
BO
1
90
100
110
Time (days)
(g)
120
130
20
15
Dicktoo
10
5
0
251
m
45
61
)1111111ki
?w
55
65
m
m
Time (days)
Heading(field)
45
fil155f0111111j1
65
75
B5
Time (day)
40
30
20
Dicktoo
Morex
10
0
85
Time (days)
220
rr 111
230
240
250
Time (days)
260
270
Figure 1: Frequency distributions for heading date under three
photoperiod regimes of 100 doubled haploid lines derived from
the cross of Dicktoo x Morex.
1
(a)
Winter Survival
(c)
30
70
60
50
40
Final Leaf Number(24h,vernalized)
(e) Maximum Tiller Nurnber(24h.vernaltzed)
30
25
Morex J
20
lDicktoo
15
30
20
10
1
Dicktoo
0
0
5
20 30 40 50 60 70 80 90 100
Montana Field Survival (%)
10
(b)
6
70
30
60
25
50
12
10
1.4
2
Number of leaves
(d)
Winter Survival
8
Final Leaf Number(24h.unvernalized)
20
20
80
40
80
Oregon Field Survival(%)
100
22
26
30
I Morex
X
10
6
8
liff-0171,
10
12
14
Number of leaves
16
5
0
2
6
10
14
15
22
Number of tillers
26
30
Field Growth Habit
(g)
30
25
20
Morex
15
Dicktoo
10
5
0
2
4
6
Grade(1-10)
10
34
(f) Maximum Tiller Number(24h, unvernalized)
35
30
25
20
15
Dicktoo
5
10
18
40
15^
30
14
Number of tillers
20
40
10
6
Figure 2: Frequency distributions for winter survival, final leaf
number, maximum tiller number, and field growth habit of 100
doubled haploid lines derived from the cross of Dicktoo x Morex.
34
12
segregants were quite different in the various vernalization and photoperiod regimes,
indicating environmental mediation of gene expression. Under field conditions, the time
from planting to heading in the DH population averaged 252 days, with a standard error
of 1.2 days, substantially longer than in any of the controlled environment tests (Figure
1g). This may be attributable to a low temperature-mediated reduction in growth rate
during the winter months. Under field conditions, there was limited phenotypic
transgressive segregation.
In the controlled environment tests, population means and ranges for heading date
were strongly affected by photoperiod and, to some extent, by vernalization. In terms of
mean performance, vernalization had little effect in the 8 h experiments (Figures la and
lb): the average heading date was 108 ± 1.4 days with vernalization and 111 ± 1.6
days without vernalization. Genotypes varied in their response to vernalization. For
example, vernalization reduced the time to heading for Morex by 19 days and by 8 days
for Dicktoo. Under 16 h light, the time to heading was dramatically reduced, as
compared to the 8 h experiments (Figures lc and 1d). Vernalization at 16 h light had a
modest effect on shortening the time to heading. The population mean was 41 ± 0.9
days with vernalization and 51 ± 1.1 days without vernalization. Vernalized Morex
headed 9 days earlier and vernalized Dicktoo 10 days earlier. In contrast to the 8 h
experiments, there was substantial transgressive segregation for heading date.
Approximately 50 % of the DH lines were earlier than Morex in both vernalized and
unvernalized treatments and 25 % of the DH lines were later than Dicktoo in the
unvernalized treatment. Under 24 h light, the population means were dramatically
13
reduced, as compared to the 8 h experiments, and there were significant numbers of
transgressive segregants (Figures le and 10. Standard errors for the unvernalized and
vernalized experiments were ± 1.5 and 1.1 days, respectively. Compared to the 16 h
experiments, the heading dates of the two parents were more similar. Differences among
the progeny were accentuated, particularly without vernalization. In general, genotypes
that were early under 16 h light were even earlier under 24 h light, and genotypes that
were late under 16 h light were even later under 24 h light. Vernalization had little effect
and in some cases even delayed heading. For example, vernalized Morex was 5 days
later but vernalized Dicktoo was 3 days earlier.
Vernalization had little effect on final leaf number under 24 h light, and parental
values were similar. The number of leaves on the main stem ranged from 6 to 16, with
a standard error of ± 0.5 leaves in the unvernalized DH population (Figure 2d).
Unvernalized Morex and Dicktoo had 7 and 9 leaves, respectively. With vernalization
the leaf number ranged from 7 to 14 in the DH population, with a standard error of ±
0.6 leaves (Figure 2c). Vernalized Morex and Dicktoo had 9 and 10 leaves, respectively.
Likewise, vernalization had little effect on maximum tiller number (Figures 2e and 20,
but Dicktoo consistently had more tillers than Morex. Ranges for maximum tiller number
in the unvernalized and vernalized experiments were 2
34 and 1
26, respectively.
Standard errors in the unvernalized and vernalized experiments were ± 1.5 and ± 1.8
tillers, respectively. Morex had 6 tillers with and without vernalization, while Dicktoo
had 10 tillers with vernalization and 12 tillers without vernalization.
14
The frequency distributions for main stem height at 3, 4, 6, 8, and 10 weeks after
transplanting are presented in Figures 3a
3j. At 3 weeks, there were only modest
differences between lines and population distributions were similar with and without
vernalization (Figures 3a and 3b). Standard errors of the unvernalized and vernalized
means were ± 0.5 and ± 0.7 cm, respectively. At 4 weeks, there was a greater range
of expression (Figures 3c and 3d), with unvernalized and vernalized standard errors of
± 0.4 and 0.7 cm, respectively. At this stage the parents were among the shortest
genotypes. By 6 weeks Morex was among the tallest genotypes, Dicktoo was
intermediate in height, and a large number of lines were shorter than Dicktoo. Standard
errors of the unvernalized and vernalized treatments were ± 0.9 and ± 1.2 cm,
respectively. By 8 weeks, approximately 25% of the population was shorter than the
population mean standard errors of the unvernalized and vernalized means were ± 1.1
and ± 1.8 cm, respectively and Morex remained among the tallest genotypes (Figures
3g and 3h). By 10 weeks only the latest genotypes were showing an increase in the main
stem height (Figures 3i and 3j). Standard errors for the unvernalized and vernalized
experiments were ± 1.3 and ± 1.5 cm, respectively.
The matrix of phenotypic correlations among traits is shown in Table 1. As
reported by Hayes et al. (1993a) the correlation between winter survival in the two field
tests was 0.57. Correlations between the two measures of winter survival and all other
traits were consistently positive and of similar magnitude, with the exception of growth
habit. More erect, spring habit genotypes tended to be those showing lowest survival.
Correlations between the vernalized and unvernalized treatments within a photoperiod
15
(a) Main Stem Height(24Mvernalized.3-week)
(b)
Main Stem Height(24h.unvernallzed 3-week)
,o
55
50
45
40
35
3
25
2
15
10
5
25
15
55
45
35
75
65
25
15
Height (cm)
(c)
35
45
55
Height (cm)
65
75
(d) Main Stem Height(24h.unvernalized.4-week)
Main Stem Height(24h.vernalized.4-week)
30
30
Dicktoo
25
25
Morex
20
20
Dicktoo
15
15
Morex
X
10
1
5
5
25
15
35
45
65
55
75
Height (cm)
Main Stem Height(24h.vemalized.6-week)
(e)
5
rill
I
15
25
35
45
Height (cm)
55
65
'75
(f) Main Stem Height(24h.unvernalized.6-week)
30
25
[ Morex
20
X
I
15
10
Dicktoo
5
0
5
15
25
35
45
Height (cm)
55
65
75
15
25
35
45
Height (cm)
55
65
75
Figure 3: Frequency distributions for main stem height of 100 doubled haploid
lines derived from the cross of Dicktoo x Morex.
16
(g)
(1) Main Stem Heinht(24h.anvernalizeci.8-week)
Main Stem Height(24h.vernalizec3.8-week)
15
25
35
45
Height (cm)
(i)
55
65
75
5
25
35
45
Height (cm)
Main Stem Height(24h.vernalized.10-week)
(j)
65
55
75
Main Stem Height(24h.unvernalized 10-week)
30
35
25
20
Mo ex
15
30
25
x
20
Dickto!
15
Morex
10
5
15
25
35
11
45
Height (cm)
55
65
75
5
1151
25
35
45
Height (cm)
55
65
Figure 3(cont.): Frequency distributions for main stem height of 100 doubled
haploid lines from the cross of Dicktoo x Morex.
Table 1: Phenotypic correlations among heading date, growth habit characters, and winter survival for 100 doubled haploid
(DH) lines derived from the cross of Dicktoo x Morex.
HDt HDt HD
8hv
HD8huv
HD16hv
HD16huv
HD24hv
HD24huv
HD Field
Leaf no.24hv
Leaf no.24huv
Tiller no.24hv
Tiller no.24huv
GHF'
SURMt
SUROt
.81**
.29**
.19
.19
.12
.66**
.24*
.07
.28**
.14
-.43**
.10
.51**
8huv
.31**
.27**
.34**
.22*
.76**
.36**
.18
.37**
.19
-.52"
16hv
.72**
.66**
.60**
.27**
.69**
.53**
.60**
.56**
SURM = winter survival in Montana
SURO = winter survival in Oregon
v = vernalized
uv = unvernalized
significant at p 0.05
0.01
.85**
.88**
.85**
.21*
.21*
.06
.88**
.86**
.91**
.82**
.82**
.87**
.94**
.20
.78 **
.17
.78"
.76
.73
.73"
.05
.24*
.85**
.78**
.75
.71**
.82**
.73**
-.40** -.47** -.41** -.38** -.66** -.39** -.37** -.41** -.43**
.54** -.61**
.43** .61** .46** .55** .37** .55** .54** .39**
.37** -.62**
.59** .41" .50** .44** .45** .58** .51** .43** .40**
.23*
t HD = heading date
GHF = growth habit in field
** significant at p
HD
Leaf Leaf Tiller Tiller GHF SURM
HD HD HD
no.
no.
no.
16huv 24hv 24huv Field no.
24hv 24huv 24hv 24huv
.57**
18
regime were consistently high and positive. Heading date responses under 16 h and 24
h light were positively correlated, but there was little relationship with heading date
under 8 h light. Heading date under 8 h light had the highly correlation with heading date
of field grown plants, confirming the important role of daylength in determining heading
date of winter barley under field conditions at 45° N latitude. Leaf and tiller numbers
were highly correlated with heading date under 24 h light. Mainstem height at the five
sampling dates was positively correlated with heading date (data not shown).
Map construction
Seventy-eight markers were used to construct a 74-point linkage map presented
in Figure 4. The assignment of linkage groups to chromosomes was based on the large
number of markers in common with previously published maps (Heun et al. ,1991;
Graner et al., 1992; Kleinhofs et al., 1993). Marker orders and distance are generally
comparable with the published maps. The map, however, provides only partial genome
coverage. Distances between linkage groups on chromosomes 1, 2, 3, and 5 were greater
than the 30 cM threshold. These segments are shown in their assumed orientation. The
highest resolution was achieved on chromosomes 2, 4, 5, and 7. The map of the long
arm of chromosome 7 has been extended and greater resolution has been achieved in the
key interval between mR and BCD265b since the report of Hayes et al.(1993a). Two
Dehydrin loci
Dhnl and Dhn2
co-segregated and were mapped within the critical
interval reported by Hayes et a. (1993a). Two additional markers Dhn4a and ABG391-
ABC167
ABG380
1
3.0
BCD175
7.0
A608
ABC158
25.0
Br z
ABC465
ABC311
ABC156
CR3
ABC170
2
5.0
30
5.0
11.0
Dhn6
BCD265a
WG1026b
ap1920
22
ABG460
a pGIu2
ABG4
CD064
ABC454
25.0
5.7
12.0
2.0
4
ABG176
5.3
4.0
5
ABG466
8.4
Hor B
ABC306
ABC468
8.2
20.0
13.7
HOrC
Hon 2
Gpi
ABG387
WG223
20 6
0.0
apVVG110s
21,0
24,4
1Est4
W5645
Ica 1
Amy l
ABG366
mHs
7.0
11.0
12.0
12.1
BCO298
ABC302
1.0
mS
WG364b
5.0
Nar 7
6.4
19.0
6.1
8.3
5.0
13.0
ABG452
CRH2
a pA DH
ABG1
000588
BcDaio
32.0
4.0
ABC174
16.1
20.2
apNar7L
9.7
16.8
13.1
ABC461
5.0
2.2
7.0
15.0
ABC305
ABG378
6
apPA
5.0
ABG472
ABC 455
CRH1
ap321
17.0
Bmyl 2
13.6
23.5
HorD
Dhn4b
iPgd
7.7
B0D265c
DhnS
Dhn3c
mR
10.0
1.0
4.6
Dhni
Dhn2
1.3 0
ap327
Figure 4: Linkage map based on 100 doubled haploid
lines derived from the cross of Dicktoo x Morex
(units are percent recombination).
19.8
apHr th
ABG373
BCD265b
Dhn4 a
1
13.3
ABC 391
20
were mapped distal to this region. The low temperature-induced clone
Cr3
mapped
to the short arm of chromosome 2.
Quantitative Trait Loci
The large QTL effect for cold tolerance on chromosome 7 reported by Hayes et
al. (1993a) was substantiated with the greater density and resolution of the current map.
Despite the presence of phenotypic transgressive segregants in both test environments
(Figures 2a and 2b), this remains the only region of the genome in which a QTL effect
for winter survival was detected. As shown in Table 3, in Montana the winter survival
LOD score for the QTL peak defined by Dhnl
BCD265b was 27.1 and this interval
accounted for more than 79 % of the variation for winter survival in this population. In
Oregon, the LOD score was reduced to 8.1, the QTL peak position shifted to the
upstream interval (mR
WG364b), and this QTL effect had a r2 of 0.31. In both data
sets, QTL LOD scores declined at the upstream marker WG364b and the downstream
marker BCD265b, while three peaks of approximately equal magnitude were seen in the
intervening intervals. There were only modest differences between peaks in terms of
maximum LOD and r2, so selection of any particular peak as the location of the
underlying gene is arbitrary. Multiple adjacent peaks may represent the effects of linked
QTLs (Martinez and Curnow ,1992), a "shadow" effect due to the linkage of adjacent
intervals with the true location of the QTL, or they may simply be a consequence of
random errors in phenotyping. Based on the available data, we can conclude that QTL
effects underlying the same phenotype measured in very different environments'map to
21
the long arm of chromosome 7. Determination of the exact position of the gene or genes
detected by these QTL effects will require analysis in alternative genetic stocks, such as
near-isogenic lines, that will allow for higher resolution mapping.
QTLs for heading date under 16 and 24 h light, for both with and without
vernalization, were detected on chromosomes 2 and 7 (Table 2). Morex contributed the
later maturity QTL on chromosome 2, while Dicktoo contributed the later maturity allele
on chromosome 7. The LOD peaks for the chromosome 2 and chromosome 7 intervals
were defined in all treatments by the ABC170 CD064 and Dhnl-BCD265b intervals,
respectively. The interval on chromosome 7 is the same as that defining winter survival
in Montana. There was variation in the magnitude of the QTL effects at these two
intervals in these four data sets. With vernalization, the effects of the QTLs at the two
intervals were similar in the two photoperiod regimes, although the effects were more
pronounced under continuous light. For example, the chromosome 2 and 7 interval LOD
scores for the 16hv were 6.4 and 5.1 and the LOD scores for the 24hv were 7.4 and 7.4.
Without vernalization, effects were again larger under continuous light, but under both
photoperiod regimes the chromosome 7 effect was larger than the chromosome 2 effect.
Under 16 h light, the chromosome 2 and 7 interval allele weights were 12.0 and 15.6
days, respectively. The multi-locus r2 values for these effects supported the differential
achieved in trait expression without vernalization and with continuous light: the
respective r2 values for the 16hv, 24hv, 16huv, and 24huv experiments were 0.48, 0.65,
0.79, and 0.83. Takahashi and Yasuda (1970) based their genetic analysis of growth habit
on heading date under 24 h light without vernalization in order to maximize differences
22
Table 2: QTL peak effects for heading date of 100 doubled haploid (DH) lines derived
from the cross of Dicktoo x Morex.
Trait
HD8hvt
Chromosome
1
3
Multi locus
HD8huvt
Multilocus
HD16hv
Multilocus
HD16huv
Multilocus
HD24hv
Multilocus
HD24huv
Multilocus
HD Field
5
5
1
3
5
5
7
2
7
2
7
2
7
2
7
1
2
3
5
5
Multilocus
7
Marker
interval
AB Cl 58-Brz
AB G4-ABC1 74
iPgd-BCD265c
Hord-ABG452
ABC] 5 8-Brz
ABG4-ABC174
iPgd-BCD265c
Hord-ABG452
mS-WG364b
ABC1 70-CD 064
Dhnl -BCD265b
ABC1 70-CD 064
Dhnl -BCD265b
ABC] 70-CD 064
Dhnl -BCD265b
ABC1 70-CD 064
Dhn1-BCD265b
ABC158-Brz
ABC1 70-CD 064
AB G4-ABC1 74
iPgd-BCD265c
Hord-ABG452
mS-WG364b
recombination
25.0
20.0
7.7
19.0
25.0
20.4
7.7
19.0
23.0
12.0
13.0
12.0
13.0
12.0
13.0
12.0
13.0
25.0
12.0
20.4
7.7
19.0
23.0
LOD
r2
weight
3.0
2.4
.16
.13
.40
.19
10.9D1
10.1
3.8
16.6
.59
2.8
3.6
6.8
3.8
2.2
.17
.22
.30
.18
.10
17.8
.64
6.4
5.1
.29
.22
13.0
.48
12.7
6.6
.30
.49
28.3
.79
7.4
7.4
.34
.34
18.8
.65
8.5
10.9
.36
.44
32.2
.83
3.3
3.0
2.1
.18
.15
.11
.20
4.7
2.1
4.1
18.2
9.7D
17.2D
11.8D
.11
.17
8.8D
9.7D
11.5D
8.8D
6.6D
7.8M
7.0D
12.0M
15.6D
16.1M
16.3D
16.4M
18.5D
7.3D
6.7D
5.9D
7.8D
5.7D
7.2D
.66
t HD = heading date; v = vernalized; uv = unvernalized.
t Letter suffix indicates parent contributing higher value allele, where D = Dicktoo and M =
Morex.
23
in trait expression. In terms of vernalization response, however, differential expression
was maximized under 16 h light. As measured by the magnitude of QTL effects, the
chromosome 7 QTL was more vernalization responsive than the chromosome 2 QTL.
For example, the differences in allele effects for the chromosome 2 and 7 QTLs without
and with vernalization under 16 h light were 4.2, 8.0 days, respectively. Under
continuous light, these same differences were only 0.3 and 2.2 days, respectively.
Under 8 h light, heading date was determined by a different set of QTLs than
those controlling responses at 16 h and 24 h light. As shown in Table 3, QTL effects
were found on chromosomes 1, 3, 5, and 7. In all cases, Dicktoo contributed the later
allele. Since Dicktoo was among the latest heading genotypes under 8 h light, these loci
may be those governing sensitivity to short daylength. A QTL on chromosome 7, defined
by the mR WG364b interval, the same interval defining the Oregon field survival QTL,
was detected at a low LOD (2.2) only in the unvernalized experiment. The QTL was
poorly resolved and had a confidence interval spanning the Dhnl- BCD265b interval.
It may represent a unique QTL or it may be an example of peak shift and thus represent
an effect of the same gene or genes determining heading date under 16 and 24 h light.
Otherwise, QTL expression was essentially the same with and without vernalization. The
QTL with the single largest effect was defined by the iPgd
BCD265c interval on
chromosome 5.
Heading date under field conditions was controlled by the same QTLs determining
heading date under all three controlled photoperiod regimes. The magnitudes of the
chromosome 1, 3, 5, and 7 QTLs were similar under 8h light and under field conditions.
24
Table 3: QTL peak effects for growth traits, field growth habit, and winter survival of
100 doubled haploid lines derived from the cross of Dicktoo x Morex.
Trait
Chromosome
Marker
interval
recombination
%
LOD
r2
weight
SURMt
7
Dhn 1 -BCD265b
13.0
27.1
.79
47DT
SUROt
7
WG364b-mR
23.0
8.1
.31
34D
Leaf no.24hvt
2
7
ABC] 70-CD 064
Dhn 1 -BCD265b
12.0
13.0
8.9
9.6
24.5
.40
2
7
ABC170-CD064
Dhnl -BCD265b
12.0
13.0
7.1
10.9
25.9
.32
.44
.76
3.3M
4.0D
2
7
ABG8-ABC3 1 1
3.0
4.6
5.3
10.0
.20
.23
.39
4.1M
4.4D
2
7
ABG8-ABC3 1 1
Dhnl -BCD265b
3.0
13.0
3.9
8.0
12.6
.16
.33
.46
5.3M
7.6D
5
Hord-ABG452
mR-Dhnl
19.0
10.0
5.5
.27
2.1M
2.7M
Multilocus
Leaf no.24huvt
Multilocus
Tiller no.24hv
Multilocus
Tiller no.24huv
Multilocus
GHFt
Multilocus
7
Dhnl -BCD265b
13.0
11.4
18.0
.41
.76
.43
.62
2.3M
2.4D
t SURM = winter survival in Montana
SURO = winter survival in Oregon
GHF = growth habit field
v = vernalized
uv = unvernalized
T Letter suffix indicates parent contributing higher value allele, where D = Dicktoo and M =
Morex.
25
The ABC170 CD064 QTL on chromosome 2 had a lesser effect under field conditions
than it did under 16h or 24h light, and there was a change in allele phase. In the
controlled environment tests, Morex contributed the larger value allele at this locus,
while under field conditions Dicktoo contributed larger value allele. The QTL peak on
chromosome 7 under field conditions that was detected in the 8 h experiments, not that
detected in the 16h and 24h experiments. As was discussed relative to other QTL effects
on the long arm of chromosome 7, additional analyses will be required to determine if
this peak shift is of biological significance.
A QTL affecting field growth habit was detected on chromosomes 5 (Table 3),
corresponding to the 8 h light heading date QTL (HorD ABG452). A second QTL was
detected on chromosome 7, in the mR
Dhnl interval, which lies between the two
intervals where QTLs were detected for 8h and 16 h/24h light, respectively. Together,
these two QTLs accounted for 62% of the variation in trait expression, with the QTL on
chromosome 7 making the larger contribution.
QTLs affecting mainstem leaf number and total number of tillers under 24 h light
mapped to a common interval on chromosome 7 and nearby intervals on chromosome 2.
The chromosome 7 interval (Dhnl
BCD265b) is the same previously identified for
Montana survival and heading date under 16 and 24 h light. Dicktoo contributed the
larger value QTL alleles for both traits, and as was seen in the case of heading date,
these QTLs showed a degree of vernalization response. This was more pronounced for
tiller number than for heading date. The QTL effects on chromosome 2 were mapped to
intervals located more than 10 cM apart, but confidence intervals for the two effects
26
overlapped. In both cases, Morex contributed the larger value alleles, the ABC170
CD064 interval was identified as the peak interval for heading date under 16 and 24 h
light.
QTLs for mainstem height revealed a pattern of differential QTL expression over
time (Table 4). At third and fourth weeks, the QTLs on chromosomes 2 and 7 associated
with mainstem leaf number and heading date under 16 and 24 hour light had the most
significant contribution to trait expression. As expected based on previous pattern of trait
expression, DH lines with the Dicktoo genotype contributed a larger value allele on
chromosome 2 and those with the Morex genotype had a larger value allele on
chromosome 7. Peak shift was seen on chromosome 2 at three weeks without
vernalization, but overlapping confidence intervals indicate that this may be the effect of
the QTL usually defined by the ABC170-CD064 interval. At three weeks, the
chromosome 7 locus had a larger effect, in terms of LOD, r2 and weight, than the
chromosome 2 locus, but by four weeks effects were comparable. By 6 weeks, however,
the chromosome 2 effect was not detectable and at both 6 and 8 weeks, only the
chromosome 7 effect was significant. At 10 weeks the chromosome 2 QTL was still not
detected, and the principal contribution was made by a previously undetected QTL on
chromosome 1 (ABC464
Brz and Brz
ABC158 for the vernalized and unvernalized
treatments, respectively). Support intervals for these peaks overlapped. This chromosome
1 interval was also a determinant of heading date under the 8h photoperiod regimes. The
support interval for the small effect QTL (LOD 2.0) on chromosome 7 also overlapped
with the previously detected effect in the Dhnl BCD265b interval.
27
Table 4: QTL peak effects of main stem height under 24 h light for 100 doubled haploid
(DH) lines derived from the cross of Dicktoo x Morex.
Trait
Chromosome
Marker
interval
%
LOD
r2
recombination
weight
2
7
AB Cl 70-CD 064
Dhnl -BCD265b
12.0
13.0
2.5
5.8
9.6
.11
.28
3.3Dt
2
7
Cr3 -AB Cl 70
Dhnl -BCD265b
2.0
13.0
3.8
6.8
11.8
.18
.27
.43
3.9D
5.0M
2
7
ABC] 70-CD 064
Dhnl -BCD265b
12.0
13.0
5.5
5.7
13.4
.26
.28
.34
15.8D
16.4M
2
7
ABC1 70-CD 064
Dhnl -BCD265b
12.0
13.0
7.3
8.2
20.0
.32
.34
.65
14.9D
15.6M
6-week 24hv
7
Dhnl -BCD265b
13.0
2.3
.12
9.7M
6-week 24huv
7
Dhnl -BCD265b
13.0
7.3
.34
19.7M
mR-Dhnl
23.0
3.7
.17
12.0M
3-week 24hv'
Multilocus
3-week 24huvt
Multilocus
4-week 24hv
Multilocus
4-week 24huv
Multilocus
.41
5.3M
no effect detected
8-week 24hv
8-week 24huv
7
10-week 24hv
1
Brz-ABC465
2.0
3.0
.13
5.6M
10-week 24huv
1
ABC158-Brz
25.0
25.8
3.7
2.0
5.3
.16
.12
.24
6.9M
6.0D
Multilocus
t v = vernalized
7
Dhn4a-AB G3 91
uv = unvernalized
* Letter suffix indicates parent contributing higher value allele, where D = Dicktoo and M =
Morex.
28
QTL interaction
Takahashi and Yasuda (1970) proposed that alleles at the Sh loci on chromosomes
4,5, and 7 interact in an epistatic fashion to determine spring vs. winter growth habit.
As interval mapping detects only individual QTL effects or their combined additive
effects, we analyzed our data in terms of two-way interactions among individually
significant QTL effects. As described in the Materials and Methods, subset of genotypes
that were non-recombinant between flanking markers were used to assess additive x
additive epistasis. For any two locus combination, four QTL combinations are possible.
In the subsequent discussion, these QTL genotypes are identified by the chromosome and
the parent subscript, where M = Morex and D = Dicktoo. For example, the four QTL
genotypes for 16 h heading date on chromosomes 2 and 7 are identified as 2M7M,
2M7D, 2D7M, and 2D7D.
As described above, QTLs for heading date under 16h light were mapped to
chromosomes 2 and 7 (Table 2). The order of mean heading date for the four QTL
genotypes was D7M2 (46 days) > M7M2 (43 days) > D7D2 (42 days) > M7D2 (35
days) with vernalization, and D7M2 (67 days) > D7D2 (53 days) > M7M2 (50 days)
> M7D2 (40 days) without vernalization. The results are consistent with the fmding that
Dicktoo contributed the late allele on chromosome 7 while Morex contributed the late
allele on chromosome 2. As indicated in Figure 5a, no interaction between these two
QTLs was detected. Thus effects are essentially additive. Vernalization shortened the
mean heading date of D7M2 and D7D2 genotypes by 21 days (P < 0.01) and 11 days
(P < 0.05), respectively, but had no significant effect on the M7M2 and M7D2
29
a
b
70
.E7 M2
65
65-
60-
60-
55-
5550-
70
<D2
M2 Er
45-
5045-
M2
40-
02 X.-
35-
D2
40-
D2
3530-
30
25
M7
16hv
D7
16hv
16huv
M7
16huv
24hv --+- 24hv
24huv
24huv
24huv
24huv
d
C 15
14131211-s
10-
98765
M7
24hv
D7
24hv
24huv
- 24hv
24huv
24hv
e130
125120-
115110-
105100-
95908580
M5(iPgd)
8hv
8hv
8huv
8huv
Figure 5: Two locus QTL genotype means for heading date under 8, 16, 24 h
light.
30
genotypes. This supports the conclusion based on the single locus data that the heading
date QTL on chromosome
Under
(42
24
7
was responsive to vernalization.
h light, the order of mean heading date was D7M2 (65 days) > D7D2
days) > M7M2 (41 days) >
days) >
D7D2 (41
M7D2 (31 days) with vernalization, and
D7M2 (66
days) = M7M2 (41 days) > M7D2 (30 days) without vernalization
(Figure 5b). No significant differences were found between the vernalized and the
unvernalized treatments. Contrary to the additive QTL effects under 16 hours of
photoperiod, a significant interaction (p < 0.05) was detected between the two QTLs.
The difference between the D7M2 and D7D2 QTL genotypes was significantly greater
(p < 0.01) than the difference M7M2 M7D2 genotypes
(23
days versus 10 days). The
mean difference between the D7M2 and the M7M2 genotypes was significantly greater
(p < 0.01) than the difference between the D7D2 and the M7D2 genotypes
(24 vs. 11
days, respectively). The interaction mean square in the ANOVA was also significant (p
< 0.01). Thus, the particular allelic combination M2D7 gave a non-additive effect and
accounts for the presence of phenotypic transgressive segregants.
Epistasis was also detected between D7 and M2 for leaf and tiller number under
24
h light (Figures 5c and 5d). For fmal leaf number, the mean difference between the
D7M2 and D7D2 genotypes was significantly greater (p < 0.05) than the difference
between the M7M2 and the M7D2 genotypes
(2.7
leaves vs.
1.7
leaves with vernalization
and 6.0 leaves vs. 1.8 leaves without vernalization). The mean difference between the
D7M2 and the M7M2 genotypes was also significantly greater (p < 0.05) than the
difference between the D7D2 and the M7D2 genotypes
(2.6
leaves vs. 1.8 leaves with
31
vernalization and 6.0 leaves vs. 1.9 leaves without vernalization). A comparable pattern
was seen for maximum tiller number. The Morex and Dicktoo alleles on chromosomes
2 and 7 thus interact in an epistatic fashion to determine fmal leaf and maximum tiller
number under 24 h light.
Four QTL peaks were detected for heading date under 8h light with vernalization
and five QTL peaks for heading date under 8 h light without vernalization (Table 4).
These individual loci were likewise tested for two-locus interactions. The only significant
interaction detected was that for the two chromosome 5 intervals defined by iPgd
BCD265c and HorD ABG452, and the interaction was significant (p < 0.01) only with
vernalization. As can be seen in Figure 5e, the heading dates of the D5(iPgd)M5(HorD)
and M5(iPgd)D5(HorD) lines were 115 and 110 days, respectively, while the M5M5 and
D5D5 homozygotes were 118 and 91 days, respectively. No significant interactions were
detected between the QTLs on chromosome 5 and 7 for field growth habit.
32
DISCUSSION
Using a 39-point linkage map, Hayes et al. (1993a) found a major QTL for winter
survival in the BCD265b-mR interval on chromosome 7 in this population. With the
current 74-point map, this chromosome region is still the only site of significant QTL
effects for this trait. The multiple peaks detected in the three intervals defined by
WG364b, mR, Dhnl/Dhn2, and BCD265b and the shift in peaks across environments
hampers resolution and only places these effects within a 48.3 cM interval. The presence
of large numbers of positive phenotypic transgressive segregants in the two field tests
may simply be a consequence of the variability associated with measurement of cold
tolerance (Fowler et al. 1979). If, however, these represent genotypic transgressive
segregants, then there must be other QTLs in regions of the genome that have not been
completely mapped, or there may be epistatic interactions between the chromosome 7
QTLs and other genome regions that did not have a significant single locus effect. More
defmitive mapping of this cold tolerance effect will require alternative genetic stocks,
such as lines that are isogenic for target chromosome interval. The association of the
Dhnl and Dhn2 loci with this winter hardiness QTL is intriguing, given the commonality
of dehydration and cold stress responses (Steponkus,1980). However, confirmation of
cause and effect relationships will require further analyses. Dehydrin loci have been
mapped elsewhere in the barley genome where QTL effects for winter hardiness-related
traits have not been detected. Dhn3, Dhn4, and Dhn5 mapped to chromosome 6, Dhn 4
to chromosomes 6 and 7, and Dhn6 to chromosome 4. Of the low-temperature induced
33
clones isolated by Sutton and Kenefick (1994), only Cr3 revealed an RFLP in this
population. No QTL effects for low temperature stress tolerance were detected in the
region of this locus, which mapped to chromosome 2. A heading date effect was detected
in the interval bounded by the adjacent downstream marker, ABC170. Ottaviano et
al.(1991) reported no association between high temperature-induced genes and drought
tolerance QTL effects in maize. Further work is necessary before we can reach the same
conclusion relative to low temperature tolerance in barley.
These data confirm the earlier fmdings of Hayes et al. (1993a) regarding
coincident map location of QTL effects for cold tolerance and heading date under 24 h
light and provide additional information on the location of genes governing growth habit
and response to photoperiod and vernalization. QTLs affecting the time from planting to
heading under field condition were detected on chromosomes 1, 2, 3, 5, and 7. The
heading date QTL mapped to the BCD265c
Dhnl interval on chromosome 7 was
moderately responsive to vernalization under 16 h light.
This QTL effect may
correspond to the Sh2 gene described by Takahashi and Yasuda (1970). The accession
of Dicktoo used in these studies does not have a vernalization requirement, yet it carries
an allele on chromosome 7 that is vernalization responsive. An epistatic response was
seen in genotypes with this chromosome 7 allele and the Morex allele on chromosome
2. Takahashi and Yasuda (1970) reported that there was a multiple allelic series at the
Sh2 locus that accounted for gradations in winter growth habit. Thus, this accession of
Dicktoo may carry an allele conferring an intermediate level of vernalization response
because even in the epistatic allelic configuration without vernalization and under 16 h
34
light, the latest DH lines headed within 30 days of Dicktoo. Two heading date QTLs
were mapped to the long arm of chromosome 5 with the 8 h light treatments. However,
the relationship of these QTLs with Sh3 and the eak cannot be established. Takahashi and
Yasuda (1970) reported only a single point linkage of Sh3 with B (black caryopsis) with
a recombination percentage of 35.8. The eak locus is downstream from the B locus,
showing 11.4% recombination with trd (third outer glume). Both B and eak are on the
long arm of chromosome 5. No QTL effects were detected on chromosome 4, site of the
sh locus. Takahashi and Yasuda (1970) reported 6.3 % recombination between sh and
mHs, a common marker in the test populations. These authors reported that certain
accessions of U.S spring barley carried the winter habit allele (Sh) at this locus. Thus,
it may be that in the Dicktoo X Morex cross alleles at this locus were fixed for Sh.
Hackett et al. (1993) mapped a QTL for heading date, which they termed a Vrn locus
effect, near fl-amylase. The distance between mHs andfl-amylase in our map is 13.3 cM.
The heading date QTL on chromosome 2 was detected under 16 or 24 h
photoperiods. The heading date QTLs on chromosomes 1, 3, and 5 were detected under
8 h light. Genes affecting heading date in the field or under short daylength were also
previously reported on chromosomes 1, 2, 3, and 5 in other barley germplasm (Doney,
1961; Woodward, 1957; Gallagher et al., 1991; Frey, 1954). Again, definitive
conclusions regarding the allelism of QTL effects and these previously reported
Mendelian loci are not possible due to the lack of common markers. However, in this
population and in the Steptoe X Morex population (Hayes et al., 1993b) a heading date
QTL where Morex contributes the larger value allele was mapped to chromosome 2 in
35
the approximate of the Ea locus (Nilan, 1964).
These data confirm the overriding importance of photoperiod and vernalization
in determining phenotypic expression. Under long day conditions (16 and 24 h light)
variation in heading date was determined by QTLs on chromosomes 2 and 7. Dicktoo
contributed the late allele on chromosome 7 and Morex the late allele on chromosome
2. Independent assortment at these unlinked loci gave rise to the transgressive segregation
observed under these photoperiod conditions. Under short day conditions (8 h light)
variation for heading date was determined by QTLs on chromosomes 1, 3, 5, and 7.
Dicktoo contributed the late alleles at all loci. As a consequence, the parents were at the
extremes of the frequency distribution. Under field conditions, where the population was
exposed to a dynamic set of photoperiod and temperature conditions, all QTLs identified
under controlled environment conditions were operating. The change in allele phase at
the chromosome 2 locus shows that a particular locus can be a determinant of trait
expression but that the test environment can dictate allele value. The low probability of
recovering genotypes with late alleles at all QTL loci may explain the position of the
parents at the extremes of the frequency distribution for heading date under field
conditions.
As demonstrated in this report, epistatic interaction can occur between QTL
effects and these interactions can be environment-specific. There were significant
interactions among certain combinations of QTL alleles controlling heading date, leaf
number, and tiller number at specified photoperiods. Since additive x additive epistasis
may be a determinant of trait expression, realized marker assisted selection based on
36
single locus effects may not equate to predicted responses. Considering the potential
complexities of three-way and higher order interactions among significant QTLs,
interactions of significant QTLs with genome regions where no QTL effects are detected,
and the additional effects of changing photoperiod and temperature under field conditions,
it is apparent that QTL analyses are only a tool for understanding plant growth and
development. The extreme complexity of these processes, and the consequent danger of
basing decisions on single point estimates of gene effects, is demonstrated by the 24 h
mainstem height data. Over a ten week period, QTLs on chromosomes 1, 2, and 7 were
identified as determinants of trait expression. If, for example, the analysis were based
exclusively on the 10 week end-point, the significant contribution of the chromosome 2
QTL would not have been detected.
The QTL for vernalization response under the 16 h light and the significant
heading date effects under 16h and 24 h light mapped to the same region on chromosome
7 as the QTL for winter survival. These data support linkage rather than pleiotropy as
the basis for this relationship. Although most of the DH lines that were vernalization
responsive had better winter survival, there were exceptions. For example, DH line 10
which was 24 days earlier to head with vernalization under 24 h light, had 12 % and 32 %
winter survival in Montana and Oregon, respectively. Several DH lines had very weak
vernalization responses but good winter survival. For example, DH lines 51, 77, and 80
were only 2, 3, and 4 days earlier with vernalization, respectively. However, their
respective winter survival percentages were 45 %, 53 %, and 82 % in Montana, and 95 %,
97% , and 100% in Oregon. QTLs for leaf number, tiller number and field growth habit
37
were coincident or overlapped with the heading date QTLs detected in the three
photoperiod environments. These may represent pleiotropic effects or the effects of
adjacent linked QTLs. Further analyses based on genotypes with defined recombination
sites within the critical intervals will be required to capitalize on the approximate map
positions revealed by these analyses.
38
REFERENCES
Brule-Babel, A.L., and D.B. Fowler. 1988. Genetic control of cold hardiness and
vernalization requirement in winter wheat. Crop Sci. 28:879-884.
Cahalan, C., and C.N. Law. 1979. The genetic control of cold hardiness and
vernalization requirement in wheat. Heredity 42:125-132.
Chang, C., S.F. Kwok, A.B. Bleecker, and E.M. Meyerowitz. 1993. Arabidopsis
ethylene-response gene ETR1: Similarity of product to two-component regulators.
Sci. 262:539-544.
Chen, F.Q., and P.M. Hayes. 1989. A comparison of Hordeum bulbosum mediatedhaploid production efficiency in barley using in vitro floret and tiller culture.
Theor. Appl. Genet. 77:701-704.
Chen, F.Q., P.M. Hayes, D. Mulrooney, and A.H. Pan. 1994b. Identification and
characterization of plant calreticulin from barley (Hordeum vulgare L.) The Plant
Cell (in press)
Chen, F.Q., and D. Prehn, P.M. Hayes, D. Mulrooney, A. Corey, and H. Vivar.
1994a. Mapping genes for resistance to barley stripe rust (Puccinia striiformis f.
sp. hordei) Theor. Appl. Genet. (in press)
Close, T.J., R.D. Fenton, and F. Moonan. 1993b. A view of plant dehydrins using
antibodies specific to the carboxyl terminal peptide. Plant. Mol. Biol. 23:279-286
Doll, H., V. Haahr, and B. SOgaard. 1989. Relationship between vernalization
requirement and winter hardiness in doubled haploids of barley. Euphytica
42:209-213.
Doney, D.L. 1961. An inheritance and linkage study of barley with special emphasis on
purple pigmentation of the auricle. M. Sci. Thesis, Utah State Univ.
39
Dorm ling, I., and A.G. Gustafsson. 1969. Phytotron cultivation of early barley mutants.
Theor. Appl. Genet. 39:51-56.
Fowler, D.B., and L.V. Gusta. 1979. Selection for winterhardiness in wheat. I.
Identification of genotypic variability. Crop Sci. 19:769-772.
Frey, K.J. 1954. Inheritance and heritability of heading date in barley. Agron. J. 46:226228.
Gallagher, L.W., K.M. So liman, and H. Vivar. 1991. Interactions among loci conferring
photoperiod insensitivity for heading time in spring barley. Crop Sci. 31:256-261.
Graner, A., A. Jahoor, J. Schondelmaier, H. Siedler, K. Pi llen, G. Fischbeck, G.
Wensel, and R.G. Herrmann. 1991. Construction of an RFLP map of barley.
Theor. Appl. Genet. 83:250-256.
Hackett, C.A., R.P. Ellis, B.P. Forster, J.W. Mc Nicol, and M. Macaulay. 1992.
Statistical analysis of a linkage experiment in barley involving quantitative trait
loci for height and ear-emergence time and two genetic markers on chromosome
4. Theor. Appl. Genet. 85:120-126.
Hayes, P.M., T.K. Blake, T.H.H. Chen, S. Tragoonrung, F. Chen, A. Pan, and B. Liu.
1993a. Quantitative trait loci on barley (Hordeum vulgare) chromosome 7
associated with components of winterhardiness. Genome 36:66-71.
Hayes, P.M., B.H. Liu, S.J. Knapp, F. Chen, B. Jones, T. Blake, J. Franckowiak, D.
Rasmusson, M. Sorrells, S.E. Ullrich, D. Wesenberg, and A. Kleinhofs. 1993b.
Quantitative trait locus effects and environmental interaction in a sample of North
American barley germplasm. Theor. Appl. Genet. 87:392-401.
Heun, M. 1992. Mapping quantitative powdery mildew resistance of barley using a
restriction fragment length polymorphism map. Genome 35:1019-1025.
40
Heun, M., A.E. Kennedy, J.R. Anderson, N.L.V. Lapitan, M.E. Sorrells, and S.D.
Tanks ley. 1991. Construction of a restriction fragment length polymorphism map
for barley (Hordeum vulgare). Genome 34:437-447.
Islam, A.K.M.R., K.W. Shepherd, and D.H.B. Sparrow. 1981. Isolation and
characterization of wheat-barley chromosome addition lines. Heredity 46:161-174.
Kleinhofs, A., A. Kilian, M.A. Saghai Maroof, R.M. Biyashev, P. Hayes, F.Q. Chen,
N. Lapitan, A. Fenwick, T.K. Blake, V. Kanazin, E. Ananiev, L. Dahleen, D.
Kudrna, J. Bollinger, S.J. Knapp, B. Liu, M. Sorre lls, M. Heun, J.D.
Franckowiak, D. Hoffman, R. Skadsen, and B.J. Steffenson. 1993. A molecular,
isozyme and morphological map of the barley (Hordeum vulgare) genome. Theor.
Appl. Genet. 86:705-712.
Knapp, S.J., and W.C. Bridges. 1990. Using molecular markers to estimate quantitative
trait locus parameters: Power and genetic variances for unreplicated and replicated
progeny. Genetics 126:769-777.
Lander, E.S., and D. Botstein. 1989. Mapping Mendelian factors underlying quantitative
traits using RFLP linkage maps. Genetics 121:185-199.
Lander, E.S., P. Green, J. Abrahamson, A. Barlow, M.J. Daly, S.E. Lincoln, and L.
Newburg. 1987. MAPMARKER: An interactive computer package for
constructing primary genetic linkage maps of experimental and natural
populations. Genomics 1:174-181.
Law, C.N. 1966. The location of genetic factors affecting a quantitative character in
wheat. Genetics 53:487-498.
Lincoln, S., M. Daly, and E. Lander. 1992. Mapping genes controlling quantitative
traits with MAPMAKER/QTL 1.1. Whitehead Institute Technical Report. 2nd
edition.
Martin, G.B., S.H. Brommonschenkel, J. Chunwongse, A. Frary, M.W. Ganal, R.
Spivey, T. Wu, E.D. Earle, and S.D. Tanks ley. 1993. Map-based cloning of a
protein kinase gene conferring disease resistance in tomato. Sci. 262:1432-1435.
41
Martinez, 0., and R.N.Curnow. 1992. Estimating the locations and the sizes of the
effects of quantitative trait loci using flanking markers. Theor. Appl. Genet. 85:
480-488.
Nielsen, G., and H.B. Johansen. 1986. Proposal for the identification of barley varieties
based on the genotypes for 2 hordein and 39 isoenzyme loci of 47 reference
varieties. Euphytica 35:717-728.
Nilan, R.A. 1964. The cytology and genetics of barley. Washington State University
Press, Washington, U.S.A.
Ottaviano, E., M. Sari Gorla, E. Pe, and C. Frova. 1991. Molecular markers (RFLPs
and HSPs) for the genetics dissection of thermotolerance in maize. Theor. Appl.
Genet. 81:713-719.
Paterson, A.H., J.W. Deverna, B. Lanini, and S.D. Tanks ley. 1990. Fine mapping of
quantitative trait loci using selected overlapping recombinant chromosomes, in an
interspecific cross of tomato. Genetics 124:735-742.
Paterson, A.H., S.D. Tanks ley, and M.E. Sorrells. 1993. DNA markers in plant
improvement. Adv. in Agron. 46:39-90.
Pugs ley, A.T. 1971. A genetic analysis of the spring-winter habit of growth in wheat.
Aust. J. Agri. Res. 22:21-23.
Pugs ley, A.T. 1972. Additional genes inhibiting winter habit in wheat. Euphytica
21:547-552.
Ramage, R.T., and C.A. Suneson. 1958. A gene marker for the g chromosome of
barley. Agron. J. 50:114.
Roberts, D.W.A. 1989. Identification of loci on chromosome 5A of wheat involved in
control of cold hardiness, vernalization, leaf length, rosette growth habit, and
height of hardened plants. Genome 33:247-259.
42
Roberts, E.H., R.J. Summerfield, J.P. Cooper, and R.H. Ellis. 1988. Environmental
control of flowering in barley (Hordeum vulgare L.). I. Photoperiod limits to
longday responses, photoperiod insensitive phase and effects of low-temperature
and short-day vernalization. Annals of Botany 62:127-144.
SAS institute. 1988. SAS user's guide: Statistics. SAS Inst. Cary, NC.
Steponkus, P.J. 1980. A unified concept of stress in plants? In: Genetic engineering of
osmoregulation 1(eds. D.W. Rains, R.C. Valentine, and A. Hollaender). Plenum
Press, New York, pp.235-255.
Sutton, F., and D. G. Kenefick. 1994. Nucleotide sequence of a cDNA encoding an
elongation factor (EF-1a) from barley primary leaf. Plant physiol. (in press)
Takahashi, R., and S. Yasuda. 1970. Genetics of earliness and growth habit in barley.
In: Nilan, R.A. (ed) Barley genetics II. Proc. 2nd Int. Barley Genet. Symp.,
Washington 1970. Washington State University Press, pp 388-408.
Thomashow, M.F. 1990. Molecular genetics of cold acclimation in higher plants. Adv.
Genet. 28:99-131.
Tischner, T. 1993. Lighting for plant growth in the Martonvasar phytotron. In: The 7th
European lighting conference, 4-7 April, Heriot-Watt University, Edinburgh, UK,
pp.400-407.
Tragoonrung, S., V. Kanazin, P.M. Hayes, T.K. Blake. 1992. STS-facilitated PCR for
barley genome mapping. Theor. Appl. Genet. 84:1002-1008.
Woodward, R.W. 1957. Linkages in barley. Agron. J. 49:28-32.
Xin, Z.Y., C.N. Law, and A.J. Worland. 1988. Studies on the effects of the
vernalization responsive genes on the chromosomes of homoeologous group 5 of
wheat. In: Proc. 7th Int. Wheat Genet. Symp. Vol. 1., pp. 675-680. Institute of
Plant Science Research, Cambridge, U.K.
43
Yasuda, S., and J. Hayashi. 1980. Linkage and the effect of earliness gene eac involved
in Chinese cultivars on yield and yield components in barley. Barley Genet.
Newsl. 10:74-76.