الفهرس 
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Abstract
The present investigation was carried out at Higher Institute for Agricultural Cooperation Farm, Regwa region, Alexandria Desert Road, Beheira Governorate, Egypt (30°11′12.0′′N, 30°34′32.7′′E), during the summer seasons of 2019 and 2020, and Molecular Genetics Laboratories, Genetics Department, Faculty of Agriculture, Ain Shams University, Shoubra El-Khema, Cairo, Egypt, during June 2022. Ten experiments (nine field experiments and a molecular genetics experiment) were procedure to study the performance, stability, environmental index and gene expression under drought and normal conditions. Six-grain sorghum genotypes Dorado, Giza 15, Giza 113, Shandaweel 1, Shandaweel 6 and Hybrid 306.
Genotypes evaluated for drought tolerance under three levels of irrigation (100 %, 70 % and 40 % of evapotranspiration). Grain sorghum genotypes were tested in nine environments that is to say nine field experiments (100, 70 and 40 % of evapotranspiration) at three sowing dates 13th July in the first season (2019), while in the second season, grain sorghum genotypes were sown on two dates. The early date was on 13th July (2020) while the late date was on 15th August (2020), E1: 40 % level of irrigation in 13th July 2019. E2: 70 % level of irrigation in 13th July 2019. E3: 100 % level of irrigation in 13th July 2019. Combined July 2019: Average of irrigation levels (40, 70 and 100 %) from evapotranspiration. E4: 40 % level of irrigation in 13th July 2020. E5: 70 % level of irrigation in 13th July 2020. E6: 100 % level of irrigation in 13th July 2020. Combined July 2020: Average of irrigation levels (40, 70 and 100 %) from evapotranspiration. E7: 40 % level of irrigation in 15th August 2020. E8: 70 % level of irrigation in 15th August 2020. E9: 100 % level of irrigation in 15th August 2020. Combined August 2020: Average of irrigation levels (40, 70 and 100 %) from evapotranspiration. Combined season 2020: Average of combined July 2020 and combined August 2020. The nine separate experiments were carried out by using Randomized Complete Block Design (RCBD), one for each irrigation level treatment with three replications. Each replication content 3 rows, long of row is 11 meters, while its width is 0.7 meter. The distance between each experiment of irrigation treatment was 3 meters width.
Distance between plant hills was 20 cm and thinning was done leaving two plants hill-1. The agriculture practices were followed as recommended except irrigation treatments throughout the growing seasons. Traits studied were plant height, no. of green leaves, stem diameter, panicle length, panicle width, panicle weight 1000-grain weight and grain yield. In addition, molecular genetics experiment was execution to measure gene expression level based on a housekeeping gene to contrast its molecular-chemical status, whether up regulated or down-regulated under different conditions (control versus drought).
The obtained data in the ten experiments illustrated as following:-
1- Analysis of variance
Analysis of variance for nine environments (three irrigation levels and three dates over the two years) showed that highly significant mean squares due to dates, irrigation levels, genotypes and their interactions were detected for all studied traits. Analysis of variance for July 2019 revealed that, highly significant mean squares due to irrigation level were detected for all studied traits. Mean squares due to genotypes were highly significant for all studied traits except 1000-grain weight. Irrigation x Genotypes interaction was significant for all studied traits except stem diameter. While analysis of variance for July and August 2020 showed that, same results highly significant mean squares due to irrigation levels, genotypes and their interaction were observed for all studied traits. Combined mean squares for two dates (July and August) during season 2020 reported that, significant mean squares due to dates, irrigation levels, genotypes and their interactions were detected for all studied traits.
2- Genotypes performance
Performance of plant height as shown, Giza 15 was the tallest cultivars for all environments, dates, irrigation levels, and their combined and years except E2 and E3, Giza 113 was the tallest cultivars for E2 and E3. Performance of no. of green l as explain, Giza 15 was the best cultivar under E2, E3 and combined July 2019. Shandaweel 1 was the best cultivar under E1. Shandaweel 6 was the best cultivar under E4, E5 and E7. Hybrid 306 was the best cultivar under E4, E5, E6, E7, E8, E9, combined July 2020, combined August 2020 and combined season 2020.
Stem diameter performances reported that, Giza 15 was the best cultivar for E9, Giza 113 was the best cultivar for E6, Shandaweel 1 was the best cultivar for all environments, dates, irrigation levels, their combined and years except E1, E2, E3 and their combined. Hybrid 306 was the best cultivar for E1, E2, E3, combined July 2019 and E4. The performances of panicle length showed that, Shandaweel 6 was the best cultivar under E4.
Hybrid 306 was the best cultivar for all environments, dates, irrigation levels, years and their combined. Hybrid 306 was the best cultivar panicle width for all environments, dates, irrigation levels, their combined and years. Panicle weight performances data revealed that, Shandaweel 6 was the best cultivar under E4, E5 and E7. Hybrid 306 was the best cultivar for all environments, dates, irrigation levels, their combined and years except E4 and E7.
1000-grain weight performances showed that Giza 15 was the best cultivar under E6 and E9. Shandaweel 1 was the best cultivar for all environments, dates, irrigation levels, their combined and years except E6 and E9. Hybrid 306 was the best cultivar under E1. Grain yield plant-1 performance reported that, Shandaweel 6 was the best cultivar under E4, E7 and E8. Hybrid 306 was the best cultivar for all environments, dates, irrigation levels, their combined and years except E4.
Average of grain yield plant-1 manifested that, the best cultivar was Hybrid 306 with 76.75 g. While the second best cultivar was, Shandaweel 6 by 75.49 g. Drought tolerance index (DTI) at 40 % of irrigation levels environments showed that, the best cultivar was Shandaweel 6. While the second best cultivar was Hybrid 306. DTI at 70 % of irrigation levels environments manifested that, Shandaweel 6 was the best cultivar and the second best cultivar was Shandaweel 1. According to the combined DTI, Shandaweel 6 was the best cultivar and the best second cultivar was Hybrid 306. DTI in July 2019 reported that, Shandaweel 6 was the best cultivar at DTI 40 % of irrigation levels environment and the second best cultivar was Hybrid 306. Hybrid 306 was the best cultivar at DTI 70 % of irrigation levels environment and the second best cultivar was Shandaweel 6. Shandaweel 6 the best cultivar was at combined DTI, whilst the second best cultivar was Hybrid 306 recording 93.48. DTI in July 2020 revealed that, Shandaweel 6 was displayed the best cultivar at DTI 40 % of irrigation levels environment and the second best cultivar was Hybrid 306. Shandaweel 6 was the best cultivar was at DTI 70 % of irrigation levels environment too and the second best cultivar was Hybrid 306. Also Shandaweel 6 was displayed the best cultivar at Combined DTI, whilst the second best cultivar was Hybrid 306.
DTI in August 2020 revealed that, Shandaweel 6 displayed the best cultivar at DTI 40 % of irrigation levels environment recorded 84.69 reflecting that, this cultivar best tolerant at harsh environments. While the second best cultivar was Shandaweel 1 recording 83.31. Shandaweel 1 was the best cultivar at DTI 70 % of irrigation levels environment recorded 94.93. While the second best cultivar was Shandaweel 6 recording 94.02. The best cultivar was Shandaweel 6 at combined DTI recorded 89.36, whilst the second best cultivar was Shandaweel 1 recording 89.12.
Drought susceptibility index (DSI) at 40 % of irrigation levels environments cleared that, the best cultivar was Shandaweel 6. While the second best cultivars were Hybrid 306. DSI at 70 % of irrigation levels of environments regarded that, the best cultivar was Shandaweel 6. While the second best cultivar was Shandaweel 1. Shandaweel 6 was the best cultivar at combined DSI, while the second best cultivar was Shandaweel 1.
DSI in July 2019 showed that, DSI at 40 % of irrigation levels environment revealed that, the best cultivars was Shandaweel 6 and the second best cultivar was Hybrid 306. DSI at 70 % of irrigation levels environment reported that, the best cultivars were Shandaweel 6 and Hybrid 306. Their combined showed that, the best cultivar was Shandaweel 6 and the second best cultivar was Hybrid 306. DSI in July 2020 revealed that, the best cultivars were Shandaweel 6 at DSI 40 % of irrigation levels environment with 0.52 showed that, this cultivar best tolerant at harsh environments. While the second best cultivar was Hybrid 306 recording 0.84.
The best cultivar was Shandaweel 6 at DSI 70 % of irrigation levels environment with 0.57 reflecting that, this cultivar best tolerant at level irrigation 70 % from optimum environments. While the second best cultivar was Hybrid 306 with 0.7. The best cultivar was Shandaweel 6 at combined DSI with 0.54, whilst the second best cultivar was Hybrid 306.
DSI for August 2020 revealed that, the best cultivars was Shandaweel 6 at DSI 40 % of irrigation levels environment recorded 0.7 showed that, this cultivar best tolerant at harsh environments. While the second best cultivar was Shandaweel 1 recording 0.76. The best cultivar was Shandaweel 1 at DSI 70 % of irrigation levels environment recorded 0.55. While the second best cultivar was Shandaweel 6 with 0.64. Shandaweel 1 displayed the best cultivar at Combined DSI with 0.65, whilst the second best cultivar was Shandaweel 6 with 0.67.
Stress tolerance index (STI) at 40 % of irrigation levels environments regarded that, the best cultivar was Hybrid 306. While the second best cultivar was Shandaweel 6. STI at 70 % of irrigation levels environments showed that, the best cultivar was Hybrid 306. While the second best cultivar was Shandaweel 6. At combined STI the best cultivar was Hybrid 306, whilst the second best cultivar was Shandaweel 6. STI in July 2019 reported that, Hybrid 306 was the best cultivar at STI 40 % of irrigation levels environment and the second best cultivar was Shandaweel 6. In addition, Hybrid 306 was the best cultivar at STI 70 % of irrigation levels environment. While the second best cultivar was Shandaweel 6.
Hybrid 306 was the best cultivar at their combined too, whilst the second best cultivar was Shandaweel 6. STI in July 2020 reported that, Shandaweel 6 displayed the best cultivar at STI 40 % of irrigation levels environment with 1.55 reflecting that, this cultivar best tolerant at harsh environments. While the second best cultivar was Hybrid 306 by 1.54.
Hybrid 306 was the best cultivar at STI 70 % of irrigation levels environment with 1.74. While the second best cultivar was Shandaweel 6 by 1.65. Hybrid 306 was the best cultivar at combined STI too with 1.64, whilst the second best cultivar was Shandaweel 6 recorded 1.6. STI for August 2020 reported that, Hybrid 306 was the best cultivar at STI 40 % of irrigation levels environment with 1.56 reflecting that, this cultivar best tolerant at harsh environments. While the second best cultivar was Shandaweel 6 by 1.52. Hybrid 306 was the best cultivar also at STI 70 % of irrigation levels environment with 1.72. While the second best cultivar was Shandaweel 6 by 1.68. Hybrid 306 was the best cultivar at Combined STI too with 1.64, whilst the second best cultivar was Shandaweel 6 recorded 1.6.
Relative performance (P) at 40 % of irrigation levels environments showed that, the best cultivar was Shandaweel 6. While the second best cultivar was Hybrid 306. P at 70 % of irrigation levels environments manifested that, the best cultivars were Shandaweel 6 and Shandaweel 1.
Combined P reported that, the best cultivar was Shandaweel 6, whilst the next best cultivars were Shandaweel 1 and Hybrid 306. P in July 2019 reported that, Shandaweel 6 was the best cultivar at P 40 % of irrigation levels environment and the second best cultivar was Hybrid 306. Shandaweel 6 and Hybrid 306 were the best cultivars at P 70 % of irrigation levels environments. Shandaweel 6 was the best cultivar at their combined, whilst the next best cultivar was Hybrid 306. P in July 2020 showed that, Shandaweel 6 displayed the best cultivar at P 40 % of irrigation levels environment with 1.12. While the next best cultivars were Hybrid 306 and Shandaweel 1 with 1.04. Also Shandaweel 6 was the best cultivar at P 70 % of irrigation levels environments with 1.04. Whilst the second best cultivar was Hybrid 306 with 1.03. Shandaweel 6 was the best cultivar at Combined P too with 1.08. While the next best cultivars were Hybrid 306 and Shandaweel 1 with 1.03. P for August 2020 reported that, the best cultivar was Shandaweel 6 at P 40 % of irrigation levels environment with 1.09 and the next best cultivars were Hybrid 306 and Shandaweel 1 with 1.07.
The best cultivar was Shandaweel 1 at P 70 % of irrigation levels environments with 1.05 reflecting that, this cultivar was the best tolerant at level irrigation 70 % from optimum environments. Whilst the second best cultivar was Shandaweel 6 with 1.04. Shandaweel 1 and Shandaweel 6 were the best cultivars at Combined P with 1.06.
Geometric mean productivity (GMP) at 40 % of irrigation levels environments showed that, the best cultivar was Hybrid 306. While the second best cultivar was Shandaweel 6. GMP at 70 % of irrigation levels environments manifested that, the best cultivar was Hybrid 306. While the second best cultivar was Shandaweel 6. Combined GMP reported that, the best cultivar was Hybrid 306, whilst the second best cultivar was Shandaweel 6. GMP in July 2019 reported that, Hybrid 306 was the best cultivar at GMP 40 % of irrigation levels environment and the second best cultivar was Shandaweel 6. In addition, Hybrid 306 was the best cultivar at GMP 70 % of irrigation levels environment, Shandaweel 6 was the second best cultivar. Hybrid 306 was the best cultivar at combined GMP with 76.1, whilst the second best cultivar was Shandaweel 6. GMP in July 2020 reported that, the best cultivar was Shandaweel 6 at GMP 40 % of irrigation levels environment with 77.85 reflecting that, this cultivar best tolerant at harsh environments. While the second best cultivar was Hybrid 306 by 77.47.
The best cultivar was Hybrid 306 at GMP 70 % of irrigation levels environment with 82.51. While the second best cultivar was Shandaweel 6 by 80.32. The best cultivar was Hybrid 306 at combined GMP with 79.99, whilst the second best cultivar was Shandaweel 6 recorded 79.09. GMP for August 2020 reported that, Hybrid 306 was the best cultivar at GMP 40 % of irrigation levels environment with 76.07 and the second best cultivar was Shandaweel 6 by 74.97. Hybrid 306 also was the best cultivar at GMP 70 % of irrigation levels environment with 79.81 reflecting that, this cultivar best tolerant at level irrigation 70 % from optimum environments. While the second best cultivar was Shandaweel 6 by 78.99. Hybrid 306 displayed the best cultivar at Combined GMP too with 77.94, whilst the second best cultivar was Shandaweel 6 recorded 76.98.
Stability factor (SF) revealed that, the best cultivar was Shandaweel 6, whilst the next best cultivars were Shandaweel 1 and Hybrid 306. SF of July 2019 revealed that, the best cultivar was Shandaweel 6 and the next best cultivar was Hybrid 306. SF of July 2020 showed that, the best cultivar was Shandaweel 6 with 1.12, whilst the next best cultivars were Hybrid 306 and Shandaweel 1 with 1.21. SF for August 2020 manifested that, the best cultivar was Shandaweel 6 with 1.18, whilst the next best cultivars were Shandaweel 1 and Hybrid 306 with 1.2.
3- Stability
The stability performance of the six genotypes was studied over nine environments i.e.; two years, three irrigation levels and three sowing dates. The join regression analysis of variance indicated that;
1- The existence of substantial variability among the genotypes was detecting for all studied traits showing that, genotypic differences were highly significant for these traits.
2- The genotypes × environment interaction including environmental linear [(E) + (G x E)] were highly significant for all the studied traits, revealing that, genotypes interacted significantly with environments, evidence that, existence inconsistency for performance of genotypes across the test environments.
3- Significance of genotype x environmental interaction linear (G x E linear) was founding for all characters revealing that, genotypes interacted significantly with environments. The presence of significant environment by genotype interaction showed the inconsistency of performance of grain sorghum cultivars across the test environments.
4- The highly significant of pooled deviation for all the studied traits indicates that, these genotypes differed considerably with respect to their stability for these traits.
5- Genotype by environment (linear) and pooled deviations were significant when tested against pooled mean squares, revealing that, both linear and non-linear components accounted for genotype, by genotype x environment variance. The large significant genotype by environment variance suggests that, the component was most important in contributing to differences in performance of genotypes across the test environments. The relatively large proportion of environment variance when compared with genotype as main effect suggests the large influence of environment on performance of grain sorghum lines.
Estimates stability statistics revealed that, the regression coefficient (bi) values of plant height ranged from 0.35 for Shandaweel 1 to 2.25 for Giza 113. Deviation from regression (S2di) varied from 3.09 for Shandaweel 6 to 133.84 for Hybrid 306. The bi values of no. of green leaves ranged from 0.66 for Shandaweel 6 to 1.36 for Dorado. S2di ranged from 0.06 for Shandaweel 1 to 0.37 for Dorado. The cultivar Giza 15 performed consistently better in favorable environments because it had high mean 8.84, its bi was more than one and its S2di closely from 0. While hybrids Shandaweel 1, Shandaweel 6 and Hybrid 306 were relatively better in stressed environments because their means were bigger than the general mean 8.51, the bi were closely from unity and the S2di were insignificant from zero. bi values for stem diameter ranged from 0.68 for Shandaweel 1 to 1.55 for Giza 15. S2di varied from 0.005 for Giza 113 to 0.03 for Giza 15.
The cultivars Giza 113 and Hybrid 306 were classified as stable, because their bi were very close to unity, S2di was very close to zero and their means were high 2.79 and 2.87, respectively. On the other hand, the cultivars Giza 113 and Hybrid 306 relatively better in favorable and stressed environments, respectively. While hybrid Shandaweel 1 was relatively better in stressed environments, because it had high mean 3.02, bi was less than one (bi < 1.0) and S2di insignificant from zero. bi values for panicle length ranged from 0.75 for Shandaweel 1 to 1.51 for Dorado. S2di values ranged from 0.35 for Shandaweel 6 to 2.46 for Shandaweel 1. Also the hybrid Shandaweel 6 was classified as stable, because its bi was very close to unity (b=1), lowest S2di and its mean was high 34.26. As well as Shandaweel 6 performed consistently better in stressed environments that, its bi was less than one (bi < 1.0). While Hybrid 306 was performed consistently better in favorable environments because it had high mean and the bi was more than one.
bi values for panicle width ranged from 0.46 for Shandaweel 1 to 1.61 for Giza 15. S2di values ranged from 0.01 for Dorado and Shandaweel 1 to 0.07 for Giza 15. The hybrids Shandaweel 6 and Hybrid 306 were relatively better in stressed environments that, their means are high, bi was less than one and S2di was insignificant from zero. bi values for panicle weight ranged from 0.83 for Shandaweel 6 to 1.23 for Giza 113. S2di values ranged from 4.21 for Hybrid 306 to 46.63 for Shandaweel 6. bi values for 1000-grain weight ranged from 0.6 for Shandaweel 6 to 1.34 for Giza 15 and Giza 113.
S2di values ranged from 0.28 for Giza 15 to 1.67 for Shandaweel 1. The cultivar Giza 15 was performed consistently better in favorable environments because it had high mean 28, its bi was more than one and its S2di was nearly from 0. On the opposite way, Shandaweel 6 was relatively better in stressed environments because its mean 27.84 bigger than the general mean 27.53, the bi was less than one and the S2di was nearly from zero. bi values for grain yield plant-1 ranged from 0.74 for Shandaweel 1 to 1.24 for Giza 15. S2di values ranged from 0.08 for Dorado to 2.58 for Shandaweel 1.
The estimates of environmental index showed that, E6 was the best performing environment for all studied traits. In addition, E3 was the best performing environment for plant height. The environment E7 was the poorest in stem diameter, panicle length, Panicle width, 1000-grain weight and grain yield plant-1. While E1 was the poorest in plant height, no. of green leaves and panicle weight. This variation in the environmental index showed that, the performance of the genotypes varied from irrigation system to another, from planting date to another planting date and from year to year.
4- Gene expression responses to drought
Gene expression quantification is a widely applied molecular genetics technique, to measure gene expression level based on a housekeeping gene to contrast its molecular-chemical status, whether up regulated or down-regulated. Additionally, when contrasting genotypes are used, qPCR can distinguish relative gene expression between both genotypes, and the same genotype under different conditions (control versus treatment). In the current analysis, the latter case was applied, two drought-sensitive varieties, along with two drought-tolerant hybrids that, showed a contrasting tolerance level in the field under drought conditions. Two genes were measured and identified as transcription factors, known as DREB2 and CBF4. Both genes were the previously reported as molecular markers for a biotic stress tolerance. The collected fresh leaves subjected to DNA and RNA extraction and isolation. A total of 4 samples, 01 and 02 for Hybrid 306 and Shandaweel 6, representing the drought-tolerant genotypes, and 03 and 04 for Dorado and Giza 113, representing the drought-sensitive genotypes, all were successful extracted. The DNA and RNA showed sufficient quality on the agarose gel, where a single dense band observed in all samples for DNA, and several ribosomal bands observed in all samples for RNA.
PCR amplification
This step performed to ensure the effectiveness of the applied primers for amplification and to optimize the annealing temperature of each gene to avoid mismatches or multiple band amplification. The genotype Hybrid 306 used as a sample to perform the PCR optimization step. The PCR was successful for the two primer pairs (i.e., two genes) and both pairs effectively bind to the DNA resulting in a single amplified band.
Real-time PCR (qPCR)
RNA was converted into cDNA by reverse transcription to quantitatively analyze the gene expression (qPCR) of the DREB2 and CBF4 genes for all tested genotypes along with the ubiquitin as a house-keeping gene as an indicator to know the extent of the change in the gene expression of the drought tolerance capacity. The quantitative gene expression analysis was successful for all triplicates of each genotype. After the real-time PCR run, the obtained CT values used to estimate ∆CT and the fold change of gene expression of DREB2 and CBF4 using subtractive equations. By extracting the CT values for all the triplicated samples, the CT values were observed between 19 and 21 for the Ubiquitin gene (HKG; as an indicator) and ranged from 20 to 23 for the DREB2 and/or CBF4 transcription factors. To define the changes in CT values (∆CT), the CT values of the transcription factors were subtracted from the CT values of the HKG independently. The ∆CT values ranged from -4 to zero, where each ∆CT was negatively powered to number 2 (i.e., anticipated duplication of DNA strand by PCR) to estimate the relative rate of change in the gene expression (fold change) for each replicate. The fold change average of the triplicates calculated to represent each genotype per quantified genes. In the DREB2 case, the greatest average fold change was 10.7 for the tolerant genotype Hybrid 306 and 9.3 for the tolerant genotype Shandaweel 6. Both genotypes were significantly higher than the average fold change calculated for the sensitive genotype Dorado and Giza 113. In the case of CBF4, the average fold change was between 2.0 and 2.7, with no significant difference between the four genotypes.
Conclusion
Shandaweel 6 and Hybrid 306 classified as drought tolerant cultivars because they have high performance and stability under drought stress and normal conditions. Shandaweel 6 and Hybrid 306 possess resistance genes for drought and other abiotic stresses, these hybrids can be use in breeding programs to improve sorghum productivity under drought stress. Stability analysis manifested that Giza 15 performed consistently better in favorable environments for 1000-grain weight, Shandaweel 6 was classify as stable for panicle length, performed consistently better in stressed environments for panicle length, panicle width and 1000-grain weight, Hybrid 306 performed consistently better in favorable environments for panicle length, performed consistently better in stressed environments for panicle width. According this results Shandaweel 6 is best genotype under stressed environments for yield attributes. Understanding the relationship among testing environments is important if plant breeders are to target germplasm better adapted to different production environments or regions. The results of environmental index showed that E6 (100% level irrigation in 13th July 2020) was the best performing environment for all studied traits. Also E3 (100% level irrigation in 13th July 2019) was the best performing environment for plant height. The environment E7 (40% level irrigation in 15th August 2020) was the poorest in stem diameter, panicle length, Panicle width, 1000-grain weight and grain yield, while E1 (40% level irrigation in 13th July 2019) was the poorest in plant height, no. of green leaves and panicle weight. This variation in the environmental index showed that the performance of the genotypes varied from irrigation system to another, from planting date to another planting date and from year to year.