Agriculture Reference
In-Depth Information
Individual
Seedling ID
Fire
Blight
Site
a
Rainfall
b
δ
13
C
c
Scab
d
Juiciness Surface Russet
GMAL3975.k
6
250 mm
-27.09 ppt
R
R
dry
0
GMAL3685.e
6
250 mm
-29.30 ppt
R
S
dry
10%
GMAL3623.f
9
450 mm
-26.33 ppt
S
S
moderate
1%
GMAL4455
4
800 mm
-26.30 ppt
R
R
medium
20%
a
Geographical location of populations in Kazakhstan.
b
Annual rainfall; source Forsline et al. (2003)
c
ppt = parts per thousand; differences of 0.5 ppt are significant.
d
R = resistant, S = susceptible
Table 3.
Example of phenotypic diversity of select lines from
M. sieversii
Kazakhstan populations
clonally propagated for simulated drought experiments where photosynthesis and stomatal
conductance were determined and roots, bark and leaves were collected for gene expression
analysis. We duplicated these studies using 'Royal Gala', a relatively drought tolerant variety
for comparison [85].
Methods Used:
We used standard methods for the morphological and physiological meas‐
urements. For the drought experiments, young trees (~1 m tall) of 'Royal Gala' propagated by
shoot proliferation were grown for several weeks in a controlled environment with standard
light and temperature conditions [44]. A simulated moderate-severe drought were imposed
by withholding water until the pots reached 40% of full saturation and maintained for 2 weeks
at this level after which the trees were sampled. A parallel control group was grown under the
same conditions, but watered to full capacity every other day. Samples from roots, bark and
leaves (fully expanded) were taken and quickly immersed in liquid N
2
. Roots were washed
for 5 min in room temperature tap water, blotted dry and placed in liquid N
2
. Bark was
removed by scraping the outer layers (down to the xylem) directly into liquid nitrogen. All
samples were stored at -80°C until use. Bark was lyophilized prior to storage at -80°C.
Total RNA was isolated, cDNA prepared and SSH performed using the protocol reported by
Bassett et al. [86] for peach. For gene analysis, we designed primers for several genes shown
to be associated with dehydration responsiveness in apple [44; manuscript submitted]. Each
primer pair was quality tested and used to prime RT-qPCR reactions in order to quantitate
gene expression in different tissues. The qPCR reactions were conducted using a kit containing
all reagents (Life Technologies, Applied Biosystems, Grand Island, NY) and the reaction
parameters were as follows: 95°C 5 min, followed by 35 cycles of 95°C 1 min, 60-65°C 1 min,
72°C 1 min and a final extension of 72°C for 10 min. Primers for a translation elongation factor
(TEF2) was used as an internal control for the qPCR experiments [87]. The relative standard
curve method was used to analyze the data.
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