Real-time PCR can quantify cDNA by amplifying energy using a threshold cycle that continues to resist. Real-time PCR programs to detect different types of stresses in resistance-specific resistance-specific sensitivity and specificity programs. In addition, real-time PCR performance requires test protocol performance by measuring sensitivity, specificity, accuracy, and reproducibility. Approved real-time PCR is an easy, fast, and accurate way to monitor the results of diagnosis and treatment in a stressful environment[10]. Real-time PCR can analyze hundreds of samples in a day[11]. The results of analysis of variance tables of CT[1]s obtained from the analysis of real-time PCR data showed a significant difference at the level of 1% between the studied lines (Table 3). The results of comparing the threshold cycles of drought-sensitive and tolerant lines in soybean showed that the pyramid line had the highest gene expression and showed a significant difference at the level of 1% with other lines. This reported that the pyramid line is also resistant to living stresses[12]. Williams, Columbus, and Douglas lines did not differ significantly in the expression of drought resistance gene, these lines had the highest expression of GR gene to drought stress after pyramid line, called semi-drought resistance lines. The lowest expression of the GR gene and the most sensitive to drought stress will line. Although tiffin, Xiaowuyie, and Amcor89 lines did not differ significantly in GR gene expression, these lines showed high sensitivity to drought stress (Figure 2). In the study of drought stress resistance and sensitivity using cluster analysis and using Euclidean distances, it can be concluded that Pace and will lines were placed in a separate cluster. The pyramid line, which had the highest expression of GR gene in drought resistance expression, was placed in a cluster with s39-99, Columbus, and PI475822a lines. Lines in a cluster with the pyramid line have the genetic potential to increase drought stress resistance. The Amcor89 and xiaowuyie lines were in a separate cluster, and the will, Douglas, and Crowford lines were in a separate cluster. The Euclidean distance diagram (Figure 3) for future crossings in improving and increasing the expression of the GR gene is forward-looking, showing the best location for subsequent crossings by the dashed line. The results of the plot matrix for comparing the first and second threshold cycles (CT1 and CT2) according to the formula 2-∆∆CT showed that the most significant difference in the first and second threshold cycles is related to the Dare line. On the other hand, the high expression results of the Pyramid line gene start in the threshold 22 cycles, while in the threshold cycle, the Dare line starts in the 30th cycle. Comparison of GR gene expression in comparing the first and second threshold cycles (CT1 and CT2) showed that GR gene expression was strongly reduced in Crowford and Will lines. In comparison, the expression of the GR gene in the CT1 and CT3 threshold cycles of the Douglas line was decreasing, but in the CT3 threshold cycle, this gene expression was increasing. The results of comparing the GR gene expression threshold cycle in the second threshold cycle showed that they would line acted out of expression, meaning that due to the lack of GR gene expression in the will line, the real-time PCR device could not calculate this amount of gene expression (Figure 4). Comparing the second and third threshold cycles in GR gene expression in the 22-line will cycle again showed the lowest gene expression. The lines that are expressed earlier are indeed the lines that feel the drought stress sooner and have to release their genes and enzymes sooner, but this gene expression and stress identification must continue while lines like tiffin, Amcore and, Xiaowuyie had earlier gene expression, but this gene expression is not continuous, so it is observed in stress-sensitive cells. Clearance of free radicals in cells by resistance genes Clearing them is acceptable when gene expression continues until stress is relieved. Lines that express their stress resistance genes earlier are not the reason for their stable resistance, but a line should have both fast and high gene expression and continuous expression of that gene. Resistance in lines is acceptable when the expression of amino acids and resistance-building proteins is done to the end of stress relief. Accelerated gene expression is not a sufficient reason for resistance to drought stress. Sometimes some lines have a high rate of gene expression, but it is observed that the cell does not show sufficient gene expression, and the line will not be sensitive to stress, or the cell will lose its vital activity due to stress and will disappear. Compared to the first and third threshold cycles (Figure 5), the odel line had the fastest expression in cycle 20, but the result of continued expression of the GR gene was again decreasing. As mentioned, a line can be called resistant that continues to express its stability against drought stress. The lines that have their threshold changes compared in Figure 6 and are outside the standard line of incremental gene expression are the lines that have decreased threshold changes. The will line had the lowest expression with a decreasing trend compared to the second and third thresholds. In environments where water is limited, genetic improvement of a crop for drought resistance is an economically attractive option [13]. However, despite the extensive resources committed to soybean breeding, progress in improving drought resistance has been slow for several reasons. Identifying lines with the highest yield potential under optimum moisture conditions is an important selection criterion in soybean. Conversely, evaluating lines from low-yielding environments under drought conditions is often not considered, because small yield differences among lines do not separate high yielding genotypes from low yielding genotypes [14]. Historically, the emphasis in soybean breeding was upon resistance to biotic stress rather than abiotic stress such as drought, due to the complexity of trait evaluation. This unfortunately, resulted in a narrow genetic base for initiating drought resistance breeding programs [15]
Table 1
Drought stress lines for determination of drought stress resistance using Glutathione Reductase gene expression
|
LINE NAME
|
Pace
|
Dare
|
odell
|
Prohio
|
LINE NAME
|
S39-99
|
crowford
|
Douglas
|
tiffin
|
LINE NAME
|
pella
|
PI 475822 A
|
Columbus
|
will
|
LINE NAME
|
Williams
|
Amcor 89
|
Xiao Wuyie
|
pyramid
|
Table 2
Primer characteristics used and reference gene
|
M.W
g/mol
|
%GC
|
T.m
|
Primer size
|
N.N
|
Primer sequence
|
Primer name
|
5733.8
|
63.16
|
62.36
|
216
|
19
|
GCGCCCGAGTCACTCATCA
|
F
|
Glutathione Reductase
|
7268.8
|
50
|
64.06
|
24
|
GCGACCCAACCAAATCACAGTCAA
|
R
|
6381.2
|
47.6
|
53
|
93
|
21
|
CTAATGGCAATTGCAGCTCTC
|
F
|
FBOX
Glyma12g05510
|
6599.3
|
47.6
|
56
|
21
|
AGATAGGGAAATGGTGCAGGT
|
R
|
Forward: F
Rivers: R
Molecular Weight:M.W
Number of Nucleotides:N.N
|
Table 3
Analysis of variance for CTs obtained from PCR real-time analysis
|
S.O.V
|
DF
|
SS
|
MS
|
Line
|
15
|
461.122
|
30.741**
|
Error
|
32
|
58.627
|
|
Total
|
47
|
|
|
** significant 1%
|