Rice (Oryza sativa L) is one of the most fundamental cereal crops, which consumed by more than half of the world's population especially in developing countries1. It is grown in different agro-ecological situations, including rain-fed areas where drought stress is common owing to variable rainfall4. In Egypt, rice is a strategic and essential crop regarding food security and considered the second important cereal crop after wheat as a mean food for the Egyptian people. Egypt at the present time is the Middle East's largest rice producer and it is considered one of the most important exportable crops, rice is produced mainly in the lower valley of the Nile River and is planted as a summer crop under irrigated conditions8. Despite, being the populations increase the most important determinant factor for the development of rice productivity to meet the increasing demand for rice a few years ago, nowadays, climate change and global warming may make the situation worse. Climate change and global warming, which affects the frequency and magnitude of hydrological oscillations, is a serious danger to agriculture, particularly in developing countries, and creates a variety of biotic and abiotic stresses for plants11,14,45. Among the abiotic factors that have shaped and continue shaping plant evolution in general and rice crop in particular, Water scarcity is the most imperative and main limitation for rice growth and production in rainfed ecosystems45–46. Water deficit is considered a natural phenomenon and is commonly defined as a period without significant rainfall (shortage of water), resulting in extensive damage to crops, and an immense loss of yield more than 70% compared to the yield under normal conditions47. When plants are exposed to abiotic stresses such as drought, several physiological, biochemical, and molecular reactions happen quickly to enable these plants to survive under these adverse conditions depending on relevant factors such as genotype, stress severity, and developmental stage. Consequently, physiological indices (such as proline content, activity of Catalase (CAT), Ascorbate peroxidase (APX) and super oxide dismutase (SOD)) related to abiotic stress can be used as a rapid and accurate method for evaluating plant tolerance to drought stress14,17–18,48. To develop the drought tolerant varieties, it is necessary to know how plants handle with drought stress issues and develop varieties that could produce high yields at a range of soil moisture conditions that actually occur in farmers’ rice fields49. In present study, we evaluated eight different rice varieties under water shortage condition for their resistance capacity through physiological, biochemical and yield approaches, in addition to determining the functional expression of some drought tolerance related genes in the most highly candidate tolerant varieties in order to identify the most desirable varieties for rice breeding programs for water shortage conditions.
According to the combined analysis of variance data, all the studied traits showed highly significant mean squares due to the environments indicating a huge difference in the performance of the studied rice varieties from well- watered conditions to water stress conditions. For varieties mean squares, all the studied traits showed the highly significant differences indicating large diversity among the used varieties to normal and water deficit conditions for all studied traits; this means that the germplasm utilized in the study possessed significant genetic diversity. The interaction between the varieties and the environments was highly significant for all studied traits; which means that the varieties under investigation behaved differently from one environment to another and can ranked differently in each environment, furthermore the non-significant interaction between the varieties and the years which was found in all the studied traits, except Chlorophyll a and Total Chlorophyll content, indicating that the tested materials performed independently from the seasonal changes hence we can recommend the superior lines. The interaction among the varieties × years × environments were non-significant for all studied characters which reflect a non-changing performance for the rice varieties in each environment in different years. As a result, the varieties studied can improve grain yield and other studied traits in water deficit-prone crops, similar results in other studies with different varieties reported by Freeg et al.,; Sitaresmi et al., and Padmashree et al., 8,50−51.
The mean performance values of the variables studied changed depending on genotype and irrigation situation. For all evaluated attributes, the highest mean values are preferred. With the exception of proline content and antioxidant enzyme activity (CAT, APX and SOD), which dramatically increased, water deficit induced significant reductions in all characters under study when compared to normal irrigation treatment. A lack of water restricts water uptake from the root system to the leaves52. As a result, it reduces water-holding capacity and stomatal movement, limiting chlorophyll synthesis, CO2 influx to leaves, and photosynthesis53–54. Drought stress depressed RWC values in all varieties, the varieties Sakha107, Hispagran and Puebla were the best able to hold a high level of water in its leaf tissues. Water scarcity stress causes stomatal closure, limiting CO2 fixation and reducing NADP+ regeneration through the Calvin Cycle55. By increasing electron leakage to molecular oxygen, these adverse circumstances increase the rate of reactive oxygen species (ROS) such as H2O2 (hydrogen peroxide), O2−(superoxide), O2 (singlet oxygen), and OH− (hydroxyl) radicals. ROS production in plants has been discovered to be enhanced by a range of environmental stresses56. ROS causes lipid peroxidation, protein denaturation, DNA mutation, and other forms of cellular oxidative damage57. Furthermore, an increase in ROS levels promotes chlorophyll breakdown, chloroplast chloroplast, and a reduction in photosystem II activity48,58. Lower water content caused a significant drop in turgor pressure, forcing stomata to close and the size of stomatal holes to shrink, resulting in decreased gas exchange and photosynthetic suppression. Drought has a typical detrimental influence on photosynthetic pigment production; hence, several studies have demonstrated a dramatic drop in chlorophyll content in drought stressed plants when compared to well-watered circumstances59–60. To deal with ROS-induced oxidative stress, plants have an effective antioxidant (enzymatic and non-enzymatic) defence mechanism61. A characteristic reaction to drought stress is the rapid accumulation of free proline. Many plants accumulate large levels of proline when subjected to drought stress in the soil, in some instances several times the total of all the other amino acids62. It is widely recognized that proline content plays an important function as an anti-oxidative defence molecule in preventing cells from damage caused by water deficit stress2. A higher level of proline content was observed in in drought- resistant varieties compared to moderately drought tolerant and drought sensitive varieties63. Many researches have shown that proline content can be utilized to screen and select drought tolerance varieties in rice2. The regulations activities of CAT, APX and SOD enzymes provide a quick and effective reaction to control the excess of ROS generated by environmental stresses64. An excess of reducing power during the water stress state, with the consequent increases in H2O2 and other ROS concentrations, is likely to cause changes in the regulation of various antioxidant enzymes. The antioxidant enzymes have a variety of yet complimentary roles in the cell defence mechanism, including direct ROS scavenging65. Yield traits are the end results of physiological processes that occur at various stages of development. Water stress severely impair the plant growth and development, usually plants reduce or stops its growth as a survival technique under water stress conditions45. It is well documented that drought causes severe reduction in rice grain yield, numbers of panicles per plant, panicle weight and 1000-grain weight59,66. Drought at booting and flowering stages negatively affect flowering and grain filling hence a high level of spikelet sterility is commonly observed under dehydration stress conditions45,67. As a result, for all studied traits, the varieties that gave the lowest reduction under water-deficit were more tolerant to water deficit than others. Thus, based on the per se performance the varieties, which gave the highest mean performance for all traits under water deficit condition, have been estimated as superior varieties and they might be useful for the incorporation of the respective traits in breeding programme for water shortage tolerance. Results are in good agreement with those reported by Al Azzawi et al.,; Abd El-Aty et al., and Herwibawa et al.,14,48,68.
The assessment of genetic diversity is crucial for the future rice breeding programs and the conservation of genetic resources. In the present study, two PCR-based molecular marker systems ISSR and SCoT were applied to assess the genetic diversity among the studied rice varieties. Both SCoT and ISSR are dominant markers. However, ISSR has randomly amplified markers, whereas SCoT is gene-targeted marker69. ISSR and SCoT markers have shown usefulness in genetic diversity studies due to their high repeatability and efficiency in detecting polymorphism70–73. The results showed that the both molecular markers exhibited comparable genetic diversity values but a higher level of polymorphism was represented by ISSR. This indicates the high efficiency of both markers in discriminating the tested varieties. The increased percentage of polymorphism noticed in ISSR markers could be explained by the greater number of ISSR primers utilized in this investigation. The efficiency of a molecular marker in genetic diversity analysis is also determined by the number of polymorphic bands per primer74. In this study, ISSR markers showed a higher number of polymorphic bands (6.6) than that of SCoT marker (6.0), indicating higher efficacy of the ISSR markers in the analysis of the studied varieties. In accordance to our results ISSR markers produced higher polymorphic bands per primer than SCoT markers in wheat and laurel varieties74–75.
The polymorphism information content (PIC) demonstrates the informativeness and discriminating power of the marker among the tested varieties. PIC values for dominant marker such as ISSR and SCoT are at maximum of 0.576. The average PIC value of ISSR primers (0.39) was found to be slightly higher than those of SCoT primers (0.33). (Botstein et al.,) stated that markers with a PIC value ranging from 0.25 to 0.50 contain useful information for genetic diversity studies77. The relatively high PIC values observed in this investigation demonstrated the informativeness and discriminative capacity of both markers system. The average PIC value obtained in this study was comparable to that found by Al-daej et al., using ISSR, and by Patidar et al. using SCoT markers in rice germplasm71,78.
The resolving power (Rp) is another factor used to measure the ability of primers to distinguish between varieties40. The average Rp of ISSR primers (6.08) was higher than SCoT primers (5.44). Moreover, the average of marker index (MI) values, which can be considered to be a common measure of efficiency in discovering polymorphism, was found to be higher in ISSR system79. Considering all the results, based on the PIC, Rp and MI values, ISSR marker was more effective than SCoT marker in the assessment of genetic diversity among the evaluated rice varieties. Our findings are in agreement with Etminan et al. and Yilmaz and Ciftci who demonstrated the usefulness of ISSR markers for estimation of genetic diversity and fingerprinting of varieties more than other DNA marker systems75,80.
The average genetic similarity obtained by ISSR and SCoT, and their combined data was 0.72, 0.74 and 0.72, respectively. This indicates that the genetic diversity of the varieties is similar when SCoT, and ISSR markers are used. Moreover, it was evident from the results that the existence of substantial genetic diversity among the tested varieties, which could be considered for parental selection in the future rice breeding programs. Interestingly, the lowest genetic similarity was observed between the local cultivar Sakha 108 and the exotic varieties Puebla and Hispagran. On the other hand, the highest genetic similarity was observed between the two Egyptian rice cultivars Sakha101 and Sakha105. These results demonstrate that geographic distance is one of the primary causes of genetic differentiation among varieties by preventing gene transmission. The large genetic distance between local and exotic varieties detected in this study, suggesting the opportunity to use these varieties in rice breeding programs for developing high-yielding and heterotic hybrids.
The results of UPGMA cluster analysis based on two genetic marker systems grouped the eight varieties into two main clusters, which generally agreed with their origin. Both ISSR and SCoT markers showed kind of similarity in the topology of their respective dendrograms. However, some differences in the subclades and the position of some varieties were observed. This variation in the number of sub-clusters obtained from the both markers could be due to the ability of each marker to divergent target regions of the genome, which in turn resulted to existence of some varieties in separate groups instead of aligning with their respective larger groups. It is clear from the cluster analysis that the exotic varieties Hispagran and Puebla from California (USA) were always clustered in the same group. This result suggested that these rice cultivars are genetically related to each other. It is possible that they may have originated from the same ancestor. Moreover, the local rice cultivars Sakha 101, Sakha 105, and Sakha 107 were placed together in all dendrograms. This observation suggested that these rice cultivars are closely related. These results were in agreement of that Mansoory et al., and Yilmaz and Ciftci who found ISSR and SCoT markers grouped the varieties according to their geographic origin75,81.