الفهرس 
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Abstract
SUMMARY
This study was carried out in the laboratories of the Genetics Dept., Fac. of Agric., Ain Shams Univ. and Ain Shams Center for Genetic Engineering and Biotechnology (ACGEB) during the period from 2016 to 2021.
Bread wheat is the most important, cultivated and consumed cereals crop in most countries worldwide, including Egypt. However, salinity is one of the major problems in wheat production which severely limits and threatens its production. Therefore, salt tolerance is an important feature that must be improved in wheat genotypes.
This study aimed at screening and evaluation of some wheat RILs for salinity tolerance, select promising lines for future breeding experiments, understand the response generated at the morphological, biochemical and molecular level following stress treatment, and validate microsatellite markers for salt tolerance.
This study was conducted to assess the effect of salinity on some bread wheat (Triticum aestivum L.) RILs and determine the salt tolerant and sensitive lines by using NaCl to simulate salinity with concentration of 102 mM and tap water as a control which equivalent 10 mM. The experiment was carried out in a growth chamber following a completely randomized design (CRD) with three replications for each treatment. Treatments included wheat cultivars Shandaweel-1 (a local salt tolerant cultivar) and Giza-168 (salt sensitive cultivar) as parents for 12 RILs that were produced via single seed descent (SSD), all genotypes were grown under two levels of salinity (10, 102 mM). The 12 RILs and their parents were grown in normal conditions (10 mM) for 25 days, then treated with NaCl salt for 30 days. We measured salt injury index (SII) for the two parents and their RILs after growing under salt stress (102 mM) for 30 days and under control conditions (10mM), in order to confine the proportional damage for NaCl. Three phenotypic traits were measured under the control and salt stress conditions namely; plant height (cm), number of tillers/plant, and number of leaves/plant. Proline content was estimated for the two parents and the studied RILs under control and salt stress conditions. The genomic DNA of the 12 RILs and their parents were extracted. To evaluate salt tolerance of the 12 studied RILs at the molecular level, 12 SSRs primers pairs, associated with salt tolerance, were tested. The twelve SSRs primers were used first to assess the two contrasting parents, and then the polymorphic markers were used to screen the studied RILs.
The results were as follows:-
1- After the plants had been kept at 102 mM NaCl for 30 days, the effects of salt stress on plant morphology were generally photographed and recorded. It was very clear that the 12 RILs were divided into two groups, each included six RILs. The first group (RIL1, RIL5, RIL9, RIL10, RIL11 and RIL12) showed ability to tolerate salt stress more than the second group (RIL2, RIL3, RIL4, RIL6, RIL7 and RIL8).
2- Plant growth was determined by plant height, number of tillers/plant and number of leaves/plant. Highly significant variations were observed between the two parents regard to the three investigated phenotypic traits. Moreover, the results of phenotypic response of wheat RILs to salinity stress indicated the varied genotypic responses. Therefore, the RILs were classified into two groups, each represented of six lines, where (RIL1, RIL5, RIL9, RIL10, RIL11and RIL12) showed high performance regard to the three measured phenotypic traits and they represented the higher group, the other six lines (RIL2, RIL3, RIL4, RIL6, RIL7 and RIL8) represented the lower group regard to their phenotypic performance under salt stress.
3- The salt injury index (SII) was calculated for the 12 RILs and their parents. Comparing with the mean value of SII values (28.11%), there was obvious difference in the salt injury index between the two parents due to NaCl treatment, where SII value of Shandaweel-1 (salt-tolerant parent P1) was lower than that of Giza-168 (salt-sensitive parent P2), indicating that the salt tolerance of (P1) was higher than that of (P2). Moreover, SII values of the highest RILs group varied from a range of (19.28% for RIL1 to 14.26% for RIL10). However, SII values of the lowest RILs group varied from a range of (38.60% for RIL4 to 32.35% for RIL6).
4- Salt tolerance trait index (STTI) for values of Shandaweel-1 were (90.64, 93.02 and 92.85%), at 102 mM NaCl stress, which were obviously higher than that of Giza-168 (75, 62.50 and 67.85 %) for plant height, number of tillers/plant and number of leaves/plant, respectively. These notable differences indicate that the salt tolerance of (P1) was higher than that of (P2). Difference between the range of STTI for the studied RILs was divided into two equal groups, namely, salt tolerant (STTI ≥ 89.20, 82.16 and 76.45%) for plant height, number of tillers/plant and number of leaves/plant, respectively, and that group represented the highest group of RILs which included (RIL1, RIL5, RIL9, RIL10, RIL11and RIL12) and its values ranged from 94.73 to 98% for plant height, 93.02 to 100% for number of tillers/plant and 93.33 to 100% for number of leaves/plant. The second group of RILs could be considered as salt sensitive (STTI ≤ 89.20, 82.16 and 76.45%) for plant height, number of tillers/plant and number of leaves/plant, respectively, and that group represented the lowest group of RILs which included (RIL2, RIL3, RIL4, RIL6, RIL7 and RIL8) and its values ranged from 77.46 to 89.02% for plant height, 67.44 to 82.5% for number of tillers/plant and 56.25 to 66.66% for number of leaves/plant.
5- Overall, salt-tolerant RILs showed greater STTI values regard to the three investigated phenotypic trait than sensitive RILs.
6- Large variations could be observed for proline accumulation among the 12 wheat RILs and between their parents. Salt stress resulted in proline accumulation in both parents, but much higher in Shandaweel-1. Values of proline accumulation under salt stress varied from 938.45 µg.g-I.F.W. for Shandaweel-1 (P1) to 478.34 µg.g-I.F.W. for Giza-168 (P2). Our results showed noticeable differences among the studied RILs. The highest amounts of Pro in leaves were observed for the highest RILs group, the minimum increase was 710.52 µg.g-I.F.W. for RIL10 and the maximum increase was 1322.08 µg.g-I.F.W. for RIL12. Nevertheless, the lowest amount of Pro in leaves was observed for the lowest RILs group, and ranged from 232.95 µg.g- I.F.W. for RIL6 to 506.23 µg.g- I.F.W. for RIL4.
7- According to the results, there must be a relationship between salt tolerance mechanisms and proline accumulation in wheat.
8- The 12 SSRs primer pairs were used first to evaluate the parental genotypes. The total number of generated bands was 17 bands with an average of 1.4 bands per primer; all primer pairs generated the lowest number of bands (1 band), except cfd49 marker located on chromosome 6D, wmc432 marker located on chromosome 1D and gwm88 marker located on chromosome 6B generated two bands, whereas gwm213 marker located on chromosome 5B generated the highest number of bands (3 bands). The molecular size (MS) of generated bands ranged from 100 bp which was generated by gwm55 marker to 361 bp which was generated by gwm88 marker.
9- Primers (cfd 9, cfd 18, cfd 46, cfd 49, cfd 183, wmc 18, wmc 503, gwm 626 and gwm 55) generated monomorphic bands in both parents and could not differentiate between the two contrasting parents, therefore, they were not used for further evaluation for the studied RILs. Whereas, primer wmc432 generated one monomorphic band with MS of 200 bp and one polymorphic band with MS of 295 bp in Shandaweel-1 (the tolerant parent), with 50% polymorphism; primer gwm88 generated one monomorphic band with MS of 172 bp and one polymorphic band with MS of 361 bp in Shandaweel-1 (the tolerant parent), with 50% polymorphism, while primer gwm 213 generated two monomorphic bands with MS of 134 and 183 bp and one polymorphic band with MS of 313 bp in Shandaweel-1 (the tolerant parent), with 33.33% polymorphism.
10- The three polymorphic markers that differentiated between the parental genotypes namely; wmc 432, gwm 88 and gwm 213, were used further to evaluate the studied RILs, and they successfully generated these unique specific bands that amplified in the tolerant parent only in the six highest RILs regard to the three measured phenotypic traits under salt stress conditions.
Proline data revealed that there must be a relationship between salt tolerance mechanisms and proline accumulation in wheat. So, proline is recommended to be measured as an indicator for salt tolerance under salt stress conditions.
The specific bands which generated by wmc 432, gwm 88 and gwm 213 primers in the present study could be used as useful markers for salt tolerance in wheat genotypes. However, further marker validations are still needed using additional wheat genotypes to confirm the usefulness of these markers for marker-assisted selection in wheat breeding programs.
Due to their high performance under salt stress conditions, amplifying a polymorphic band within three SSRs primers associated with salt tolerance and accumulating the highest amounts of proline content under salt stress, six RILs (RIL 1, RIL 5, RIL 9, RIL 10, RIL 11 and RIL 12) out of the 12 studied could be considered as promising materials for improving bread wheat in breeding programs in the future.