CSIRO PUBLISHING
www.publish.csiro.au/journals/ajar
Australian Journal of Agricultural Research, 2005, 56, 1069–1077
Backcross reciprocal monosomic analysis of leaf relative water content,
stomatal resistance, and carbon isotope discrimination in wheat under
pre-anthesis water-stress conditions
Shahram Mohammady-DA,C , Keith MooreB , John OllerenshawB , and Behrooz ShiranA
A Faculty
B School
of Agriculture, University of Shahrekord, Iran.
of Biology, University of Newcastle upon Tyne, NE1 7RU, UK.
C Corresponding author. Email: shfaza@hotmail.com
Abstract. Monosomic plants from an Australian variety (Oxley) having low stomatal resistance (SR), low leaf
relative water content (LRWC), and high carbon isotope discrimination () were crossed with variety Falchetto
having opposite characters in order to produce F2 backcross reciprocal monosomic families. The families were
assessed under pre-anthesis water-stress conditions in a controlled growth chamber. F2 backcross reciprocal
monosomic analysis suggested possible allelic variations between chromosomes 1A, 3A, 6A, 7A, 7B, 1D, and 4D of
Falchetto and their homologues in Oxley for LRWC. This analysis also suggested possible allelic variation between
chromosomes 5A, 1A, and 3A of Falchetto and their homologues in Oxley for SR. Extending the analysis to the
F3 disomic generation and the assessment of LRWC at this generation confirmed that reciprocals for chromosomes
3A and 6A showed significant differences. F2 backcross reciprocal monosomic analysis for suggested allelic
variations on chromosomes 1D, 4D, and 5D. However, chromosome 1D from Falchetto had the highest difference
from its homologue in Oxley. Assessing the reciprocals of this chromosome for vegetative evapotranspiration
efficiency (ETEveg ) at the F3 disomic generation indicated that the observed variation for was translated into
differences for ETEveg . These results indicate that chromosome 1D of Falchetto is promising in reducing and
that the improvement of wheat varieties for ETEveg can be done by selection for . Finally, plieotropic effects of
some chromosomes were observed for the characters under study. This suggests the existence of genetic factors
on these chromosomes affecting more than one character. However, some pleiotropic effects could also be due to
non-genetic developmental interactions.
Additional keywords: disomic, plieotropic effect, within-family variation.
Introduction
Assessment of wheat varieties for physiological components
of drought tolerance has been used as a quick technique
for screening drought-tolerant varieties (Adjei and Kirkham
1980; Blum et al. 1981; Winter et al. 1988; Gumuluru
et al. 1989; Chaves 1991; Ehdaie 1995; Dhanda and Sethi
1998; Golestani Araghi and Assad 1998). Physiological
characters of plants, particularly those related to plant
water status, have a great deal of importance in growth and
development of plants under water-stress conditions. Leaf
relative water content (LRWC), stomatal resistance (SR),
water use efficiency (WUE), and its components, stomatal
characteristics and carbon isotope discrimination ()
have been used as examples of some characters that determine
water-stress tolerance of wheat genotypes. LRWC is the
relationship between fully turgid water content and actual
water content of plant tissues when they are subjected to water
stress. Therefore, LRWC indicates the ability of plants to keep
© CSIRO 2005
their water status at a reasonable level when they experience
water stress. Chaves (1991) pointed out that LRWC represents
the water status of plants, whereas other parameters such as
water potential can be affected by soil, plant, and atmospheric
water status. Therefore, LRWC may be a more appropriate
indicator of plant water status. LRWC is also closely related
to cell volume and reflects the balance between water
supply and transpiration (Schonfeld et al. 1988). In addition,
genotypic variation for LRWC in wheat has been reported by
various investigators (Blum and Johnson 1993; Dhanda and
Sethi 1998; Mentewab and Sarrafi 1998). This variation helps
plant breeders to select for high LRWC in wheat cultivars and
landraces. Stomatal resistance (s/cm) is a character leading
to water regulation of plants. This character has been used as
a criterion to screen drought-tolerant varieties by several
researchers (Shimshi and Ephrat 1975; Adjei and Kirkham
1980; Blum et al. 1981; Jones 1987; Gumuluru et al.
1989; Golestani Araghi and Assad 1998). Golestani Araghi
10.1071/AR05038
0004-9409/05/101069
1070
Australian Journal of Agricultural Research
and Assad (1998), working on Iranian varieties, reported
that stomatal resistance was recognised as a beneficial
drought-resistance indicator. Since most of the water escapes
through the stomata (Wang and Clarke 1993), stomatal size
and frequency are among factors that influence stomatal
resistance. Carbon isotope discrimination () is a character
indicating the amount of 13 C depleted by photosynthesis
mechanisms. This character is related to drought-tolerance
indicators such as stomatal resistance and water use efficiency
(Farquhar and Richards 1984; Griffiths 1993; Taiz and Zeiger
1998). Furthermore, this character has indicated negative
association with grain yield under some drought conditions
(Richards et al. 1998).
When monosomic series are available for only one variety
of the two varieties under study, backcross reciprocal
monosomic analysis can be used to determine allelic
differences between any two homologous chromosomes
each belonging to one of the varieties. Backcross reciprocal
monosomic analysis has been used as a method to
identify chromosomes involved in various quantitative
traits (Snape and Law 1980; Snape et al. 1983; Law et al.
1987) and even resistance to fungal diseases (Buerstmayr
et al. 1999). The application of this method has been
restricted to the characters that are easily measurable under
field conditions. Some characters need to be studied under
controlled environments at a particular stage of development.
For instance, the effect of water stress is different at different
stages of growth. Thus, F2 backcross reciprocal plants
should have minimum differences in growth stage when
they are subjected to water stress. This study may be the
first attempt to use this method for detecting possible allelic
variation between two varieties for physiological traits
under growth-room conditions. Data from the previous
studies (Mohammady-D 2002) demonstrated that Oxley
showed clear differences from Falchetto for LRWC, SR,
and carbon isotope discrimination under pre-anthesis
water-stress conditions. The aim of this study was to identify
possible chromosomal variation between the two varieties
for water-stress tolerance in terms of LRWC, SR, and
carbon isotope discrimination using backcross reciprocal
monosomic analysis.
Materials and methods
Development of F2 backcross reciprocal monosomic families
Figure 1 illustrates the procedure for developing backcross reciprocal
monosomic families. All 21 Oxley monosomic lines provided by
Dr McIntosh from Australia were crossed with the variety Falchetto
in a heated greenhouse at the Close House field station of
Newcastle University, UK. Oxley monosomic lines were used as
female parents and Falchetto was used as the male parent. The
derived monosomic F1 hybrids were cytologically identified in pollen
mother-cell meiosis using the Feulgen staining method. The F1 hybrids
were backcrossed reciprocally to the initial Oxley monosomic lines
to produce reciprocal BC1 seeds. BC1 seeds were then planted
and BC1 monosomic plants were again cytologically identified
S. Mohammady-D et al.
Variety Oxley
Variety Falchetto
×
×
Selfing
×
Selfing
2n = 42
2n = 41
2n = 40
2n = 40
2n = 41
2n = 40
Fig. 1. A procedure for the development of F2 backcross reciprocal
monosomic families (only 2 pairs of homologous chromosomes are
shown). Modified from Snape et al. (1983).
for reciprocal families from 18 out of the 21 monosomic lines. The
reciprocal backcross F2 seeds were then harvested from monosomic
reciprocal BC1 hybrids and used for the present experiment carried out to
evaluate F2 backcross reciprocal monosomic families for the characters
under study.
Development of F3 backcross reciprocal disomic families
Calculating coefficient of variation (CV) using the data measured
on variety Falchetto over different experiments indicated that LRWC
and had good stability across the experiments (see Table 5). Thus,
development of the F3 generation was restricted to the chromosomes that
were involved in controlling LRWC and chromosome 1D the reciprocals
of which had indicated the highest difference for (see Table 4).
Therefore, reciprocal F3 lines were only developed for chromosomes
1A, 3A, 6A, 7A, 7B, 1D, and 4D.
To develop F3 backcross reciprocal disomic families, two F2 disomic
plants were extracted from each F2 backcross monosomic family of the
above chromosomes (except from a line carrying chromosome 4D of
Falchetto in which only one disomic plant was identified). Seeds from
each F2 plant were germinated separately to develop F3 families so that
for each F3 reciprocal family, 2 duplicates were formed. For instance,
chromosome 1A had 2 F3 disomic reciprocal families, one having
chromosome 1A from Oxley and the other chromosome 1A from
Falchetto, and each reciprocal F3 family included two duplicates, each
originated from a different F2 disomic plant.
The method for assessing F2 backcross reciprocal families
for LRWC, SR, and ∆
Backcross reciprocal lines belonging to 18 different chromosomes and
the 2 parents were assessed in a series of experiments. Experiments
had to be done in sequence due to limitation in space and in order
to provide enough time for recording the measurements. Thus, the
experiments were started with parents in the first week and continued
with reciprocal lines from one or two chromosomes approximately
Backcross reciprocal monosomic analysis in wheat
each week thereafter (in total 14 successive experiments). Five plants
from variety Falchetto were included in each experiment as controls to
monitor the changes in the characters due to alterations in the starting
times of the experiments.
Ten seeds of each parent and 30 large seeds with normal
endosperm development from each reciprocal BC1 hybrid were
germinated in an attempt to increase the number of 2n = 42 plants
(T. Worland, pers. comm.). The seedlings were planted in 5-cm pots
and were fully vernalised for 4 weeks at Close House field station
in a cold cabinet at 2◦ C min. and 7◦ C max. with an 8-h photoperiod.
At least 16 seedlings from each line were transplanted into 10-cm pots
at Moorbank Garden and were grown in a greenhouse. The artificial
light was provided in the greenhouse to lengthen the photoperiod to
16 h. Light intensity varied between 160 and 230 µm/m.s at the plant
surface due to the distance between the plants and light sources. The
pots were evenly irrigated by a wet mat provided with an automatic
water supply. The plants were grown in this situation until at least
10 plants of each reciprocal line reached Zadoks Stages 37–39
(Zadoks et al. 1974). At these stages at least 10 early plants were
selected and late plants were discarded in order to provide space
to grow the next series of experiments and to grow some of the selected
plants to produce F3 seeds. Selection for early plants was done in an
attempt to increase the proportion of disomic plants and consequently to
decrease hemizygous effects (Law et al. 1987). The selected plants were
carefully irrigated so that the soil was saturated with water and water
started to drain from the bottom of the pots. The pots were labelled
randomly and transferred to a growth room (min. 15◦ C, max. 23◦ C;
16-h photoperiod). Water stress was imposed by withholding water
for 7 days. Stomatal resistance was measured in the middle, and
LRWC of the flag leaf was measured at the end of the water-stress
period for all of the 10 plants. Carbon isotope discrimination indicated
a negative significant relationship with dry matter (DM) in an
experiment carried out by the authors (Mohammady-D 2002) and in
other studies (Ehdaie and Waines 1994, 1997; Mohammady-D 2004).
Therefore, the dried flag leaf from each plant was stored separately for
future measurement of carbon isotope discrimination in case differences
were found between reciprocals for DM production.
Stomatal resistance was measured at midday (11 a.m.–2 p.m.) on
both the adaxial and abaxial surfaces of the flag leaf, using a Delta-T
Diffusion porometer model AP4. This equipment measures stomatal
resistance based on the water vapour coming out through the leaf tissues
in a unit of seconds per centimeter (s/cm). LRWC was determined
by using the last fully expanded leaf (flag leaf) from each plant
at the end of the water-stress period. LRWC was calculated using
the equation LRWC = [(Fw − Dw)/(Tw − Dw)] × 100, where Fw is
fresh weight, Dw is dry weight, and Tw is turgid weight. The fresh
weights of leaf samples were obtained immediately after excision,
then the leaves were put into test tubes containing distilled water.
After 24 h, both adaxial and abaxial surfaces of leaves were dried
with paper towel and turgid weights were obtained. Leaf dry weights
were recorded after 48 h of oven drying at 80◦ C. For measuring ,
leaves were finely ground to a powder to ensure homogeneity and to
achieve greater accuracy in determining carbon ratio (Boutton 1991).
Only a small amount of plant material is required for most combustion
systems (Griffiths 1993); therefore, samples were weighed in amounts
of 1 ± 0.05 mg. The carbon isotope composition (δ‰) of samples was
determined using an elemental analyser isotope ratio mass spectrometer
known as ANCA-SL (automated nitrogen carbon analysis unit for
solids and liquids), PDZ Europe 20/20 mass spectrometer. was
calculated using the equation (‰) = (δa − δp)/(1 + δp), assuming
that δa = −7.6‰ (Farquhar and Richards 1984).
After measuring the characters, 5 plants of each line suspected to
be 2n = 42 on the basis of phenotype (earlier plants, normal vigour)
were grown in order to produce F3 seeds. The other 5 plants were
Australian Journal of Agricultural Research
1071
harvested and oven-dried in order to obtain an indication of vegetative
dry weight for each line. The late spikes of the selected plants were fixed
to cytologically identify disomic plants.
Assessing reciprocal families for LRWC and ETEveg
in the F3 generation
Assessment of the F3 generation was restricted to only LRWC and
ETEveg . ETEveg was used to see whether differences observed between
reciprocals for are translated to ETE. At least 18 plants from
each F3 line were grown in a greenhouse until Zadoks Stage 37–39
of the growth cycle and then 10 plants from each line were subjected
to water stress under growth-room conditions as explained in the
previous section. LRWC was measured for F3 lines belonging to the
above chromosomes. Thus, for each F3 family, data were available for
2 duplicates. The duplicates were ranked within each family according
to the performance of the F2 plants from which the F3 lines originated.
Comparisons were made between each pair of duplicates that were
located in the same position in the ranking order, in an attempt to
minimise the differences due to background variation. Student t-tests
were used to compare reciprocal families. Since the difference found
for between the F2 reciprocal lines for chromosome 1D was highly
significant, WUE was also calculated for duplicates belonging to
this chromosome in order to assess the effect of this chromosome
on ETEveg . The plants of backcross reciprocal lines for chromosome 1D
were grown in the growth room from the seedling stage and the water
used was measured for each pot. ETEveg was calculated for these
lines as described in Ehdaie (1995).
Statistical methods
Student t-test (Steel and Torrie 1976) was used to compare reciprocal
means, and the equality of within-F2 family variance was tested between
reciprocal pairs in each chromosome for LRWC, SR, and using
Leven’s test (Leven 1960). CV was calculated as the ratio of standard
deviation to the mean using the data obtained from Falchetto across
the experiments. This value was used to select a less environmentally
effective character for study in the F3 generation.
Results and discussion
F2 reciprocal monosomic analysis
The mean performance of the F2 backcross reciprocal
families for each inter-varietal homologous chromosome
comparison is shown in Table 1 for LRWC. Among
the 18 chromosomes evaluated, reciprocal families for
chromosome 1A, 3A, 6A, 7A, 7B, 1D, and 4D showed
significant differences for LRWC, chromosomes 1D and
4D from Oxley and the others from Falchetto having positive
effects on LRWC. Reciprocal families for chromosomes 1A,
5A, and 6A showed significant differences for SR. In these
lines all 3 chromosomes from Falchetto had positive effects
on SR (Table 2). The observed differences between reciprocal
families belonging to chromosomes 7B and 1D may be
due to differences in plant size measured as DM production
(Table 3). When grown in a pot, larger plants will deplete
water faster than smaller plants and consequently larger
plants experience more severe water stress than smaller
plants. This phenomenon causes more stomatal resistance
and lower LRWC. Therefore the observed difference for
these chromosomes may not be genetic but an experimental
1072
Australian Journal of Agricultural Research
S. Mohammady-D et al.
Table 1. Mean performance and standard error of each
backcross reciprocal family for LRWC (%) together with the
difference between each pair of reciprocal families and the results
of the t-test (N = 10)
Table 3. Mean performance and standard errors of each
F2 backcross reciprocal family for DM (g) together with the
difference between each pair of reciprocal families and the results
of the t-test
Chromosome
designation
Chromosome origin
Oxley
Falchetto
Difference
between
reciprocals
P value
Chromosome
designation
Chromosome origin
Oxley
Falchetto
Difference
between
reciprocals
P value
1A
3A
4A
5A
6A
7A
1B
2B
3B
4B
5B
6B
7B
1D
3D
4D
5D
6D
67.80 ± 2.76
62.90 ± 4.86
60.67 ± 3.62
74.31 ± 2.76
68.43 ± 2.91
68.93 ± 2.98
87.53 ± 1.52
64.74 ± 4.47
64.27 ± 4.26
77.03 ± 3.00
57.74 ± 6.15
79.83 ± 2.95
52.68 ± 2.96
79.88 ± 3.98
67.35 ± 3.79
69.70 ± 2.97
74.07 ± 2.58
84.64 ± 4.20
79.95 ± 4.18
79.93 ± 3.66
67.67 ± 4.55
69.28 ± 2.96
80.98 ± 3.94
80.34 ± 3.12
85.61 ± 2.12
56.61 ± 4.90
67.62 ± 5.03
69.31 ± 4.92
62.91 ± 4.49
82.90 ± 2.41
70.82 ± 2.03
67.88 ± 2.51
74.76 ± 4.34
59.59 ± 3.76
76.40 ± 3.14
86.79 ± 3.40
−12.15*
−17.03**
−7.00
5.03
−12.55*
−11.41*
1.92
8.13
−3.35
7.72
−5.17
−3.07
−18.04***
12.00*
−7.41
10.11**
−2.33
−2.15
0.02
0.01
0.24
0.23
0.02
0.02
0.47
0.23
0.62
0.20
0.51
0.39
0.0008
0.02
0.21
0.05
0.57
0.70
1A
3A
4A
5A
6A
7A
1B
2B
3B
4B
5B
6B
7B
1D
3D
4D
5D
6D
1.27 ± 0.14
1.16 ± 0.09
1.34 ± 0.08
1.16 ± 0.07
1.25 ± 0.06
0.80 ± 0.02
0.54 ± 0.06
1.22 ± 0.12
1.37 ± 0.12
0.99 ± 0.08
0.77 ± 0.05
0.91 ± 0.09
0.85 ± 0.03
0.68 ± 0.10
1.07 ± 0.08
0.98 ± 0.05
0.69 ± 0.06
0.90 ± 0.07
1.25 ± 0.12
1.23 ± 0.07
1.10 ± 0.10
1.25 ± 0.08
1.04 ± 0.09
0.75 ± 0.05
0.77 ± 0.08
1.58 ± 0.15
1.21 ± 0.14
1.17 ± 0.07
0.72 ± 0.04
0.67 ± 0.05
0.75 ± 0.02
1.29 ± 0.06
1.02 ± 0.02
1.13 ± 0.06
0.92 ± 0.06
1.28 ± 0.25
0.02
−0.07
0.24
−0.09
0.21
0.05
−0.23
−0.36
0.16
−0.18
0.05
0.24
0.10
−0.61
0.05
−0.15
−0.23
−0.38
0.90
0.52
0.09
0.45
0.33
0.13
0.04
0.10
0.42
0.10
0.53
0.06
0.04
0.0006
0.80
0.10
0.03
0.19
Euploid
parents
66.90 ± 2.74
80.50 ± 2.83
−13.60**
0.008
Euploid
parents
0.55 ± 0.04
0.72 ± 0.05
−0.17
0.04
*P < 0.05; **P < 0.01; and ***P < 0.001.
Table 2. Mean performance and standard error of each
backcross reciprocal family for SR (s/cm) together with the
difference between each pair of reciprocal families and the results
of the t-test (N = 10)
Chromosome
designation
1A
3A
4A
5A
6A
7A
1B
2B
3B
4B
5B
6B
7B
1D
3D
4D
5D
6D
Euploid
parents
Chromosome origin
Oxley
Falchetto
Difference
between
reciprocals
P value
11.83 ± 0.57
6.21 ± 1.16
5.83 ± 1.09
6.61 ± 0.70
10.76 ± 1.76
6.99 ± 1.14
8.31 ± 1.88
7.32 ± 1.45
15.75 ± 2.94
8.39 ± 0.95
10.17 ± 1.13
7.22 ± 1.46
11.95 ± 1.32
6.10 ± 1.70
8.45 ± 1.58
8.06 ± 1.69
11.58 ± 2.45
9.03 ± 1.49
16.71 ± 1.72
7.08 ± 1.29
7.61 ± 1.46
10.30 ± 1.28
16.25 ± 1.74
5.17 ± 0.94
9.95 ± 1.31
10.79 ± 2.09
13.71 ± 2.23
11.26 ± 1.65
12.60 ± 1.34
9.31 ± 1.33
11.01 ± 2.31
6.88 ± 1.33
6.73 ± 1.66
5.34 ± 1.64
15.48 ± 2.68
14.76 ± 2.55
−4.88**
−0.87
−1.78
−3.69*
−5.49*
1.82
−1.64
−3.47
2.04
−2.87
−2.43
−2.09
0.94
−0.78
1.72
2.72
−3.90
−5.73
0.005
0.89
0.24
0.02
0.04
0.25
0.48
0.19
0.59
0.15
0.18
0.30
0.73
0.72
0.46
0.26
0.30
0.07
6.58 ± 1.57
12.60 ± 1.45
6.02*
0.02
*P < 0.05; **P < 0.01.
artefact common to potted plants. A surprising result is that
chromosomes 1D and 4D from Oxley showed a positive
effect on LRWC. This event is the reverse of the difference
observed between Oxley and Falchetto. One explanation
for this result is that these characters are determined by
additive interaction between several chromosomes so that
Falchetto, having more chromosomes with a positive effect,
showed higher LRWC than Oxley. Similar results were also
reported by many researchers using different cytogenetic
methods. Buerstmayr et al. (1999) using backcross reciprocal
monosomic analysis found chromosome 2B of a variety
susceptible to Fusarium head blight (Hobbit-sib) to contribute
to resistance to this disease to a greater extent than its
homologue from the resistant variety U-136.1. In addition,
Giura and Saulescu (1996) using an F2 monosomic analysis
reported that chromosomes 4B, 5B, and 5D of a variety with
high grain weight (G603-86) had negative effects on this
character in comparison with the homologous chromosomes
from a variety with low grain weight (Favourit). Law and
Worland (1996) using substitution lines of Bezostaya into
the Cappelle-Desprez background found that chromosomes
5A, 5D, and 3B of the variety Bezostaya increased, but
chromosomes 1B, 2D, 3D, 4A, 6D, and 7A reduced the height
of Cappelle Desprez.
Carbon isotope discrimination was measured for the
reciprocals that had shown significant differences for DM.
Comparison between reciprocal lines for 18 chromosomes
revealed that reciprocals showed significant differences for
Backcross reciprocal monosomic analysis in wheat
Australian Journal of Agricultural Research
Table 4. Mean performance and standard error of 4 pairs of
F2 backcross reciprocal families for (‰) together with the
difference between each pair of reciprocal families and the results
of the t-test (N = 10)
1B
7B
1D
5D
Chromosome origin
Oxley
Falchetto
27.08 ± 0.20 26.54 ± 0.16
26.21 ± 0.13 26.05 ± 0.11
24.94 ± 0.16 23.61 ± 0.13
25.85 ± 0.30 24.93 ± 0.12
Difference
between
reciprocals
P value
1.2
1
0.8
0.6
0.4
y = –0.1715x + 5.2198
R 2 = 0.6732**
0.2
0.54*
0.16
1.33***
0.92
0.04
0.36
4.3 × 10−6
0.011
0
23
24
25
26
27
28
∆‰
Fig. 2. Relationship between DM and measured on the
mean of 8 F2 backcross reciprocal monosomic families under
water-stress conditions.
*P < 0.01; ***P < 0.001.
DM production in chromosomes 1B, 7B, 1D, and 5D
(Table 1). In this case, chromosomes 1B, 1D, and 5D from
Falchetto and chromosome 7B from Oxley had positive
effects on this character. Therefore was measured for these
reciprocals only. The results with these lines are presented
in Table 4. Among 4 chromosomes, which were evaluated
for , reciprocals for 3 chromosomes showed significant
differences for this character from which the line having
chromosome 1D from Falchetto had the highest difference
from its relevant reciprocal.
The above results for LRWC and SR have been obtained
from 14 different separate experiments and for in
4 different experiments, each experiment containing
reciprocals of one or two chromosomes. Data obtained from
variety Falchetto as a control in various experiments indicated
that the CV of SR, LRWC, and across the experiments
was 25.62, 5.38, and 2.00%, respectively (Table 5). These
results indicate that LRWC and are more stable than SR
and therefore more reliable for screening genotypes
under drought.
Table 5. Mean performance of variety Falchetto in different
experiments for SR (s/cm), LRWC (%) and (‰) (N = 5)
Experiment
1.4
DM
Chromosome
designation
1073
SR
LRWC
1
2
3
4
5
6
7
8
9
10
11
12
13
14
Mean
s.d.
14.03
10.02
10.15
15.58
7.54
11.41
11.09
12.37
8.47
10.01
10.65
12.22
19.00
9.92
11.60
2.97
79.53
70.28
79.40
70.67
78.31
73.73
69.51
77.99
79.08
78.14
70.96
75.18
69.30
73.20
74.66
4.01
CV (%)
25.62
5.38
26.58
26.03
26.24
25.34
0.52
2.00
Pleiotropic effects
As can be seen from the above results, reciprocals
for some chromosomes showed significant differences
for 2 or more characters. For example, reciprocals for
chromosomes 1A and 3A showed differences for both LRWC
and SR, reciprocal families for chromosome 7B showed
significant differences for both LRWC and DM production,
and those for chromosome 1D showed differences for LRWC,
DM, and . Reciprocals for chromosomes 1B and 5D also
produced differences for both DM and , which confirmed
the association between DM and observed in the
parents (data not shown). Similarly, a negative relationship
was also found between DM and in F2 monosomic
families (Fig. 2).
Pleiotropic effects of chromosomes can be due to
different genes for each character on the same chromosome
or due to the pleiotropic effect of one gene. Some plieotropic
effects also may be due to non-genetic developmental
interactions. Pleiotropic effects of the genes on LRWC,
SR, and DM are not surprising, because these characters
can affect each other, positively or negatively. For instance,
high stomatal resistance can lead to higher LRWC due to
suppression of transpiration. Furthermore, higher vegetative
DM can lead to higher water consumption and reduction in
LRWC when soil water is exhausted. High SR in varieties
with high vegetative growth can also help the plants to keep
water content at a reasonable level under stress conditions.
Thus a combination of SR and vegetative dry matter
can affect the amount of reduction in LRWC under preanthesis water-stress conditions. Vegetative dry matter
can affect due to an increase in stomatal resistance
during the water-stress period. However, any increases in
photosynthetic capacity can also reduce and consequently
increase DM (Ehdaie and Waines 1994; Rebetzke et al.
2002). Griffiths (1993) believes that the high capacity of
RUBISCO enzyme reduces carbon isotope discrimination
in C3 plants.
Using cytogenetic studies, pleiotropic effects of
chromosomes have been widely reported in the literature for
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Australian Journal of Agricultural Research
S. Mohammady-D et al.
various characters. Snape and Law (1980) using backcross
reciprocal monosomic analysis of chromosome 5A, involving
varieties Cappelle Desprez and Bezostaya, reported the
pleiotropic effect of this chromosome on ear emergence
time, height, grain weight per plant, and spikelet number
per ear. Snape et al. (1983) used backcross reciprocal
monosomic analysis involving monosomics 5A and 7A of
Cappelle Desprez and 2 alien substitution lines in which
chromosomes 5A and 7A of variety CS were substituted by
chromosomes 5U and 7U of Aegilops umbellulata in order
to study possible effects of chromosomes 5U and 7U on
some agronomic characters. The results indicated that both
Ae. umbellulata chromosomes reduced height relative to the
Cappelle Desprez chromosomes. They also had detrimental
effects on yield components, reducing spikelet number
and grain weight per ear. Chojecki et al. (1983) studying
reciprocal monosomic analysis between varieties CS and
Spica reported that chromosomes ID and 7D of Spica
increased both grain weight and DNA content in comparison
with their homologues in CS. Giura and Saulescu (1996)
studying all 21 F3 disomic progenies derived from crosses
between 21 monosomic lines of the Romanian cultivar
Favorit and line G603–86 reported that chromosome 1B
increased both grain length and width. Farshadfar et al.
(1995) using intervarietal single-chromosome substitution
lines reported that chromosome 5D of Cappelle Desprez
increased the LRWC and reduced the relative water loss
of CS when it was substituted in the CS background.
They also reported that chromosome 7A reduced both
relative water loss and susceptibility to drought (DS).
Some pleiotropic effects of chromosome arms have
also been reported on various agronomical characters
(Ehdaie and Waines 1997).
Variation within F2 families
Plants within each F2 backcross reciprocal monosomic
family will differ due to segregation for the remaining
20 chromosomes contributing to the genetic background.
For the crosses involving any 2 varieties, this segregation
should produce the same genetic variance between plants
within each reciprocal family because the hemizygous
chromosome does not contribute to the between-plant
segregation when there is no hemizygous effect (Snape
and Law 1980). If the reciprocal lines are not significantly
different from each other for within-family variations,
then it may be concluded that the background segregation
for both reciprocal lines is the same and the difference
that is observed between the means of the 2 reciprocal
lines is predominantly due to allelic variation between
the chromosomes involved. One method to test whether
reciprocal families for each chromosome are statistically
different for within-family variation is the heterogeneity
(equality) test (Snape and Law 1980). The equality of
within-family variance was therefore tested between
reciprocal pairs in each chromosome for LRWC, SR,
and using Leven’s test (Leven 1960). None of
the reciprocal pairs, which had shown differences for
LRWC, had statistically different within-family variances
(Table 6). This implies that the difference observed between
the means of these reciprocals is predominantly due to
allelic variation. Differences between variances of reciprocals
approached significance for chromosome 4B (P = 0.056).
Table 6. Results of Leven’s test for equality of within-family variance between each pair
of F2 backcross reciprocal monosomic families for SR, LRWC, and Chromosome
designation
1A
3A
4A
5A
6A
7A
1B
2B
3B
4B
5B
6B
7B
1D
3D
4D
5D
6D
LRWC
Test
P value
statistic
1.930
0.486
0.637
0.005
0.806
0.347
0.456
0.074
0.185
4.160
2.276
0.003
1.413
1.253
0.625
0.021
0.487
0.073
0.18
0.49
0.43
0.95
0.38
0.56
0.51
0.79
0.67
0.06
0.15
0.96
0.25
0.28
0.44
0.89
0.494
0.79
‰
SR
Test
statistic
P value
Test
statistic
P value
12.31
0.005
0.487
2.449
0.199
0.241
0.101
1.611
0.108
6.640
0.691
0.004
2.556
0.239
0.006
0.238
0.328
5.727
0.003
0.94
0.49
0.13
0.66
0.63
0.75
0.22
0.75
0.02
0.42
0.95
0.13
0.63
0.94
0.63
0.57
0.03
–
–
–
–
–
–
0.027
–
–
–
–
–
0.125
1.457
–
–
2.540
–
–
–
–
–
–
–
0.87
–
–
–
–
–
0.73
0.584
–
–
0.128
–
Backcross reciprocal monosomic analysis in wheat
Australian Journal of Agricultural Research
1075
This indicates that within-family variation (due to
background or hemizygosity effect) is possibly a reason
for no detected differences between reciprocal means for
this chromosome.
In the case of SR, the equality test for within-family
variance was also performed to test the equality of variance
between the reciprocal lines in each chromosome. Among
the chromosomes in which the means of reciprocals
had shown significant differences, only the reciprocals of
chromosome 1A had a significant difference for withinfamily variances (P < 0.01), whereas the differences between
the variances of the reciprocals for chromosomes 3A and 5A
were not statistically significant. This implies that the
difference observed between the means of reciprocals for
chromosome 1A is at least partly due to differences in
the genetic background. Reciprocals for chromosomes 4B
(P = 0.02) and 6D (P = 0.03) also had different within-family
variances, indicating the possible effect of within-family
variation masking the differences between reciprocal means
for these chromosomes.
Table 7. Mean and standard errors of F3 backcross reciprocal
disomic families (selfed from F2 disomic plants) of 7 chromosomes
(2 duplicates in each family) for LRWC (%) and the results of the
t-test (N = 10)
Backcross reciprocal F3 disomic analysis for LRWC
and ETEveg
Reciprocal pairs in 7 chromosomes showed differences
for LRWC at the F2 monosomic generation (Table 2).
Table 7 presents the mean performance of the reciprocals
in the F3 disomic generation for LRWC. Two duplicates
were assessed for each reciprocal family except for the
reciprocal line having chromosome 4D from Falchetto. For
chromosomes 3A and 6A, reciprocals were statistically
different in both duplicates. The pooled data over the
duplicates also indicated significant differences between the
reciprocals of these chromosomes (Table 7). In addition,
reciprocals for chromosome 4D also showed differences for
LRWC in the only evaluated duplicate pair and when data
were pooled over the duplicates of reciprocal families having
chromosome 4D from Oxley.
These results confirmed that the differences observed
between reciprocals of these chromosomes at the
F2 generation are possibly due to large allelic variation
between the 2 varieties, and the effect of background on the
observed differences is small. On the other hand, reciprocals
for chromosomes 1A, 7A, and 1D did not show differences
for LRWC in one of the duplicates in the F3 generation. This
indicates that the effect of background is considerable on
the observed differences in both the F2 and F3 generations
for these chromosomes and that allelic variation is not
large enough to differentiate between reciprocal duplicates
regardless of their genetic backgrounds. Reciprocals for
chromosome 7B showed significant differences in LRWC for
only one of the duplicates. This implies that the difference
observed in the reciprocal of this chromosome is not allelic
because in the F3 generation this difference was observed
neither in the second duplicate nor in the pooled data.
Chromosome
Chromosome origin
Oxley
Falchetto
Difference
P value
−2.22
−21.07**
−11.64**
0.54
0.001
0.003
1A
Dup1
Dup2
Pooled
58.66 ± 2.32 60.88 ± 2.70
53.23 ± 4.90 74.30 ± 3.20
55.95 ± 2.71 67.59 ± 2.55
3A
Dup1
Dup2
Pooled
56.83 ± 2.90 72.19 ± 3.72 −15.36**
0.003
49.67 ± 2.34 64.21 ± 2.76 −14.54*** 4.59 × 10−4
53.25 ± 1.99 68.20 ± 2.43 −14.95*** 2.84 × 10−5
6A
Dup1
Dup2
Pooled
64.02 ± 1.80 70.59 ± 1.35 −6.57**
0.007
64.20 ± 1.67 78.14 ± 1.85 −13.95*** 1.17 × 10−5
64.11 ± 1.21 74.37 ± 1.41 −10.26*** 2.64 × 10−6
7A
Dup1
Dup2
Pooled
66.04 ± 3.18 82.48 ± 1.80 −16.80*** 1.44 × 10−4
72.88 ± 2.58 78.36 ± 2.20 −5.48
0.11
69.46 ± 2.14 80.42 ± 1.46 −10.96
0.001
7B
Dup1
Dup2
Pooled
62.72 ± 3.22 73.06 ± 3.09
59.46 ± 7.50 60.56 ± 4.41
61.09 ± 4.02 66.81 ± 2.99
−10.34*
−1.10
−5.72
0.02
0.89
0.26
1D
Dup1
Dup2
Pooled
76.04 ± 3.64 79.24 ± 1.97
84.04 ± 2.00 65.09 ± 2.15
80.04 ± 2.22 72.17 ± 2.15
−3.20
18.95***
7.87**
0.42
1.78 × 10−6
0.01
4D
Dup1
Dup2
Pooled
72.77 ± 3.35 63.22 ± 2.99
70.60 ± 3.05
–
71.69 ± 2.22 63.32 ± 2.99
9.55*
–
8.37*
0.04
–
0.03
*P < 0.05; **P < 0.01; ***P < 0.001.
These inconsistencies over the generations can be due to
background variations (Snape and Law 1980) and have been
reported for other quantitative characters. Chojecki et al.
(1983) studied grain weight in the F1 reciprocal monosomic
lines and F3 disomic generations derived from the F1 lines.
In the F1 monosomic comparison, reciprocal pairs in
chromosomes 1A, 1D, 3D, 4B, and 7A showed significant
differences for grain weight. They extracted disomic plants
from the F2 monosomic reciprocal families and evaluated the
F3 disomic reciprocal families of the above chromosomes
for grain weight. In the F3 disomic comparisons, significant
differences were observed between the reciprocals for
chromosomes 1A, 1D, and 7A and the differences observed
between the reciprocals of chromosomes 3D and 4B in the
F1 monosomic generation disappeared in the F3 disomic
comparisons. This seems to be due to differences between
the genetic backgrounds of the reciprocals in the F3 disomic
generation, arising from sampling errors in using bulked
seeds from random F2 plants. However, they did not draw
1076
Australian Journal of Agricultural Research
S. Mohammady-D et al.
any conclusion about these results and they have been left
unexplained in the paper.
In the case of chromosome 1D, ETEveg was also
measured. According to the results presented in Table 8,
reciprocals for this chromosome showed significant
differences for ETEveg in both duplicates and in the pooled
data. This implies that allelic variation ETEveg is larger than
background variations so that the reciprocal pairs showed
significant differences irrespective of genetic backgrounds
of the duplicates.
Much of the genetic variation for improving stress
tolerance has been lost during selection and modern breeding
(Araus et al. 2002). Therefore, other genetic materials such as
landraces rather than modern varieties should be used to
obtain a large improvement in stress tolerance. Selected
landraces can contribute to the enhancement of wheat
production in dry regions by direct use for cultivation or by
using various methods of plant breeding in order to improve
high-yielding but drought-susceptible varieties so that they
can tolerate drought. The varieties Oxley and Falchetto can be
used as controls for screening the landraces for , SR,
and LRWC.
This study has added to the literature that
chromosomes 1D, 3A, and 6A of variety Falchetto may be
promising for improving water-stress tolerance in wheat, and
further studies including application of molecular markers
specified for these chromosomes would provide more
evidence about the role of these chromosomes in controlling
water-stress tolerance related characters. Detecting genetic
variations between varieties and landraces using cytogenetic
analyses is still a useful method and the rapid increase in
the application of molecular markers will not reduce the
importance of cytogenetic approaches in plant breeding.
Law and Worland (1996) pointed out that ‘what is required
is a fusion between the new marker techniques and both
the established and developing methods of cytogenetics.
With this regard, cytogeneticists have one prime asset in
wheat which is not the case for other crops and that is the
availability of a very large number of monosomic series and
chromosome substitution lines’. With these explanations,
using molecular methods in order to investigate various
characters in cytogenetic stocks is a new area of research
in order to determine the precise role of the chromosomes
Table 8. Mean and standard errors of F3 backcross reciprocal
disomic families of chromosome 1D (2 duplicates in each family)
for ETEveg and differences between the reciprocals
Chromosome
Dup1
Dup2
Pooled
Chromosome origin
Oxley
Falchetto
2.95 ± 0.10
2.77 ± 0.06
2.86 ± 0.06
*P < 0.05; ***P < 0.001.
3.25 ± 0.09
4.22 ± 0.14
3.73 ± 0.14
Difference
P value
−0.30*
−1.45***
−0.87***
0.04
2.03 × 10−8
1.08 × 10−6
previously reported using cytogenetic approaches and to
find molecular markers associated with the characters on the
candidate chromosomes.
Acknowledgments
We thank Prof. J. Snape and the late T. Worland,
John Innes Centre, Norwich, UK, for their valuable comments
on the experiments, and Prof. R. A. McIntosh for providing
the monosomic lines.
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Manuscript received 2 February 2005, accepted 3 August 2005
http://www.publish.csiro.au/journals/ajar