Abiotic stress is defined as any change in environmental conditions that might
reduce or adversely affect plant growth or development (Bruce
et al., 2002). Drought is a pervasive problem throughout most of the
world's cultivated regions and it has been recognized as the single most important
limitation to productivity. Previous studies indicated that prolonging irrigation
intervals led to decreased growth, yield and yield components of maize (De
Souza et al., 2009; Shiri et al., 2010;
Abdel-Latif et al., 2011). The utilization of
molecular markers, developed by analysis of Randomly Amplified Polymorphic DNA
(RAPD), for the improvement of drought tolerance is an important part of the
solution to improve global maize production. Bulked segregant analysis of F2
plants was developed by Michelmore et al. (1991)
as a simpler alternative to isogenic line analysis where the highest and lowest
extremes of the F2 population are bulked for the development of RAPD
molecular markers needed for QTLs-assisted selection. Several investigators
(Abdel-Tawab et al., 2002; Younis
et al., 2007) tried to identify molecular markers associated with drought
and salt tolerance in maize and sorghum. Therefore, this study aims to identify
molecular markers associated with maize drought tolerance via RAPD technique.
MATERIALS AND METHODS
Field experiments: The present study was conducted at the Agricultural
Research Station of Ain Shams University, Shalakan, Kalubia Governorate, Egypt,
during the three successive growing seasons of 2009, 2010 and 2011.
Two inbred lines of maize (Zea mays, L.), i.e., Sd-63 (drought tolerant)
and Gm-18 (drought sensitive), are chosen based on previous screening experiment
(Abdel-Latif et al., 2011), Seeds of the two
pure lines were obtained from Maize Res. Section, Field Crop Res. Institute,
ARC, Giza, Egypt. In 2009 growing season, The grains of the two inbred lines
were grown in the field and crossed to obtain the F1 hybrid grains.
In 2010 growing season, some of the F1 grains were sown in the field
and plants were selfed to obtain the F2 grains. In 2011 growing season,
the two maize parental lines and their respective F1 hybrids were
grown and evaluated in the field under normal and drought stress conditions.
Two separate field trails were carried out; one trail under normal irrigation
(irrigation every 13 days) and the other under drought stress (skipping the
4th and 5th irrigations through growing season). The drought stress trail involved
the two parental lines, F1 and F2 plants. Each experiment
was conducted in a randomized complete block design with three replicates to
study the effect of water stress on yield of different maize genotypes. Each
replicate consisted of 25 ridges for drought condition and 9 ridges for normal
condition. Three ridges were planted for each of P1, P2
and F1 and 16 ridges for F2 under drought condition. Three
ridges were planted from each of P1, P2 and F1
under normal irrigation. Each ridge was five meter long and 70 cm width. Planting
was done in hills spaced at 25 cm apart and hills were thinned at one plant
per hill after about 21 days from sowing. Plant height (cm), 100 kernel weight
(g) and grain yield per plant (g) were recorded on all guarded plants in the
middle row for each of the P1, P2 and F1 and all guarded
plants in all rows of F2 for each replicate. The F2 generation
was represented by 700 individual plants. The common agricultural practices
of growing maize were applied properly as recommended in the district.
Statistical analysis: The analysis of variance was performed according
to the method described by Snedecor and Cochran (1981).
Molecular genetic studies
Genomic DNA extraction: Genomic DNAs were isolated on a small scale from
200 mg of one week old etiolated seedlings of both inbred lines along with their
F1 and the two F2 extreme groups (the most drought tolerance
and the most drought sensitive group). Leaves were ground to a powder using
liquid nitrogen in Eppendorf tubes and DNA were isolated using plant genomic
DNA Mini Prep Kit (V-gene Biotechnology, china, Cat. No. 69104) according to
the manufacturer manual.
PCR conditions and electrophoresis: Six out of 20 primers for RAPD were
used in this study. Names and sequence of the selected primers are illustrated
in Table 1.
The amplification conditions and PCR mixture were set according to Williams
et al. (1990) for RAPD analysis.
To visualize the PCR products, a 15 μL of each reaction was loaded on
1.2% agarose gels. These gels were run at 90 v for 1 h, visualized with UV Transilluminator
and photographed using UVP gel documentation system (Gel Works 1 D advanced
software, UVP (Ultraviolet Products).
|| Names and sequence of the selected primers for PCR-RAPD
Data analysis: Data for each analysis was scored using the UVP gel documentation
system. Fragment sizes were estimated using 100 and 1 kb DNA standards (Bioron,
RESULTS AND DISCUSSION
Data for plant height, 100 kernel weight and grain yield per plant for the
two maize inbred lines (Sd-63 and Gm-18) with their F1 hybrid under
normal and drought condition are presented in Table 2. Significant
differences were detected between parental lines for all agronomic traits under
normal and water stress conditions indicating the variability existed between
the two maize parental lines. Abdel-Sattar and Ahmed (2004),
De Souza et al. (2009), Shiri
et al. (2010) and Abdel-Latif et al. (2011),
in their drought stress experiments on maize, indicated the high genetic potentiality
of some maize genotypes based on some agronomic traits.
The results revealed that the parental line Sd-63 was taller (with the means
of 147 and 116.67 cm under normal and drought stress, respectively), higher
100 kernel weight (with the means of 28 and 21.67 g for both treatments, respectively),
higher yielding ability (with the means of 98.33 and 69.67 g for both treatments,
respectively) and consequently more drought tolerant than the other parental
line Gm-18. F1 plants had higher performance as for plant height,
100 kernel weight and grain yield per plant and consequently more drought tolerant.
After the two selected maize inbred lines i.e., Sd-63 (tolerant) and Gm-18
(sensitive) were crossed to obtain F1 grains, some of these F1
grains were sown in the field and selfed to obtain F2 grains. Drought
experiment was conducted to investigate the response of the F2 segregating
population to water stress. F2 plants were classified in a descending
order, based on their tolerance to water stress, into groups of 30 F2
individuals in which the most four tolerant and the most four sensitive groups
(Table 3) were selected for subsequent molecular analysis.
The four most drought tolerant groups had means of 176.25 cm, 28.59 and 198.75
g for plant height, 100 kernel weight and grain yield per plant, respectively
while the four most drought sensitive groups had means of 92 cm, 18 and 72.25
g for the above mentioned traits, respectively.
Molecular genetic analysis: The bulked segregant analysis was adopted
in this study (Michelmore et al., 1991) to detect
markers for drought tolerance in maize. The bulked segregant analysis identifies
markers linked to a molecular trait of interest in the segregating F2
population generated from the hybrid between the two contrasting genotypes (tolerant
and sensitive in this study).
|| Mean performance of two maize inbred lines and their cross
for some agronomic traits under normal and drought conditions
||Mean performance of the most drought tolerance and the most
drought sensitive F2 groups with respect to some agronomic traits under
water stress conditions
||RAPD-PCR profile of (1) tolerant, (5) sensitive maize parents,
(3) tolerant F1, (2) the most tolerant F2 and (4) the most sensitive F2
plants (M = marker and RAPD = random amplified polymorphic DNA)
Two DNA bulks from the most two contrasting F2 groups were used
along with their parents and F1 plants to develop RAPD (Random Amplified
Polymorphic DNA) markers associated with water stress tolerance. The DNA bulks
of F2 of the two extreme groups, for their performance under drought
condition, F1 and their parents (Sd-63, tolerant and Gm-18, sensitive)
were tested against twenty 10-mer random primers. Data were considered for only
six out of twenty primers (Fig. 1), banding pattern for the
six primers (A01, A05, A06, A07, B08 and C02) were illustrated in Fig.
1 and scored as present (1) or absent (0) as shown in Table
4. Four out of the six primers (A01, A05, A06 and B08) were developed molecular
markers for drought tolerant as shown in Table 4.
||RAPD fragments of six RAPD primers with tolerant (1) and sensitive
maize parents (5), tolerant F1 (3), the most tolerant F2 (2) and the most
sensitive F2 (4) plants (MW = molecular weight, RAPD = random amplified
For PCR reaction with the primer A01, two universal bands at Molecular Weights
(MW) 97.54 and 71.34 bp characterized the most tolerant F2 group.
In the same time, three bands at MW 151.89, 108.24 and 92.57 bp were found in
the sensitive parent (Gm-18) and the most sensitive F2 group while
were absent in the tolerant groups.
With respect to PCR reaction with the primer A05, two bands at MW 870.90 and
458.79 bp were characterized the tolerant parent (Sd-63) and the most tolerant
F2 group while were absent in the sensitive groups.
Regarding PCR reaction with the primer A06, three universal bands at MW 766.72,
690.84 and 465.92 bp were showed to be present for the tolerant groups (the
tolerant parent Sd-63, the most tolerant F2 group and the tolerant
F1) while were absent in the sensitive groups. In the same time one
universal band at MW 439.7 bp was characterized the sensitive parent Gm-18 and
the most sensitive F2 while was absent in the tolerant groups.
With respect to PCR reaction with the primer B08, two bands at MW 812.08 and
768.2 bp were showed to be present for the tolerant groups (tolerant parent,
F2 and F1) while were absent in the sensitive groups.
Regarding the other two primers (A07 and C02), the data of these primers were
not developed any molecular markers for drought tolerant in maize.
The results of the present study are in harmony with the findings of Abdel-Tawab
et al. (2002) and Abdel-Bary et al. (2005),
they detected some RAPD markers associated with drought and salt tolerance in
maize. Rashed et al. (2006) and Younis
et al. (2007), they detected some RAPD markers associated with salt
tolerance in Sorghum.
Results of this study which contain two maize inbred lines, drought tolerant
(Sd-63) and drought sensitive (Gm-18), their F1 generation and bulks
of the two extreme F2 plant groups (the most tolerant F2
group and the most sensitive F2 group) were tested against six RAPD
primers to determine some molecular markers for drought tolerance in maize.
This analysis revealed that, four RAPD primers (A01, A05, A06 and B08) out of
six developed molecular markers for drought tolerance in maize. These RAPD markers
could be considered as reliable molecular markers associated with drought tolerance
in maize that can be utilized during breeding programs via marker-assisted selection.