الفهرس 
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Abstract
This work was carried out at the Department of Genetics, Faculty of Agriculture, Assiut University, Assiut, Egypt during 2012-2016.
The present investigation aimed to study the following objectives:
iv. To conduct an in vitro evaluation of drought tolerance in guava using PEG.
v. To assess genetic diversity among different responsive genotypes to draught stress using SRAP and ISSR techniques.
vi. To establish specific DNA markers associated with drought tolerance.
Forty guava trees grown in the experimental farm of Genetics Department, Faculty of Agriculture, Assiut University, Assiut Governorate, Egypt were used in this study as a source of explants for in vitro drought evaluation. These landraces have been collected previously from different locations in Egypt, which have exposed high level of variability at both phenotypic and molecular evaluation. Shoot pieces, 10-20 cm long, were taken from newly growing flushes (~2 months old) in March and April or September and October.
The results of in vitro drought evaluation could be summarized as follow:
1- The preliminary experiment showed that increasing PEG in the medium had a significant effect on both percentage of response (%R) and number of shoots per explant (NSE).
2- There was no significant effect of both 4 and 6% PEG on the response of explant compared with control.
3- The use of PEG at 10% was enough to inhibit the response of explants and shoot formation completely in all tested landraces.
4- The eight percent of PEG was selected as the optimal concentration for drought tolerance evaluation.
5- The analysis of variance showed highly significant differences among the tested landraces and among PEG concentrations as well as the interaction between them (Landrace × Concentration) in both %R and NSE.
6- Drought treatment caused a significant reduction in both %R and NSE associated positively with increasing levels of PEG.
7- The average percentage of reduction (%RED) of %R due to 8% PEG treatment ranged from 21.97 to 90.00% with an average of 48.30% compared with control, whilst the %RED in the NSE ranged from 4.00 to 88.11% with an average of 52.57% compared with control.
8- NSE showed a high heritability (0.94), while %R showed a moderate H2 (0.45) in the control.
9- Under drought stress H2 was increased for %R (0.71) and decreased for NSE (0.64). In addition, DSI showed a moderate H2 (0.52) under drought stress.
Molecular analysis
Genomic DNA was extracted from the highest five (i.e. L14, L15, L16, L34 and L44) and lowest five (i.e. L7, L8, L9, L18 and L38) responsive landraces, on basis of response under 8% PEG, and was used for molecular analysis. Two molecular markers namely sequence related amplified polymorphism (SRAP) and inter simple sequence repeats (ISSR) were used to discriminate among the highest and lowest responsive landraces. All extracted DNA samples were successfully amplified with both markers and data were scored and analyzed.
The results of molecular analysis could be summarized as follow:
SRAP molecular analysis
1- Out of the 10 SRAP primer combinations used in this study, five primers (Me4-Em1, Me2-Em2, Me4-Em4, Me6-Em10 and Me10-Em10) gave polymorphism and were selected for data analysis.
2- The total number of bands generated by the 5 selected primers of SRAP was 71 of which 18 (25.35%) bands were polymorphic.
3- The number of bands per primer ranged from 8 to 21 bands, with an average of 14 bands per primer.
4- The highest percentage of polymorphism (%P) showed in SRAP was 38.46% generated by primer Me4-Em1.
5- The polymorphism information content generated by SRAP primers ranged from 0.02 to 0.17 with an average of 0.09, while the average resolving power of primer was 1.92 ranged from 0.4 to 3.6 and the diversity index ranged from 0.02 to 0.32 with an average of 0.19.
6- SRAP markers used in this study were able to generate three unique bands, two of them were specific for drought tolerance and generated by Me2-Em2 and Me4-Em4, which were existed only in landraces with high response under stress and one band specific for susceptibility which was found only in low responsive landraces, generated by Me4-Em1.
7- The UPGMA cluster analysis of SRAP based on Jaccard’s coefficient of similarity was able to separate the highest five responsive landraces and the lowest five responsive ones into two main clusters separated at similarity coefficient of 0.87.
ISSR molecular analysis
1- Out of the 10 ISSR primers used in this study, five primers (UBC-840, UBC-826, UBC-834, UBC-846 and UBC-810) gave polymorphism and were selected for data analysis.
2- The total number of bands generated by the 5 selected primers of ISSR was 33 of which 14 (42.42%) bands were polymorphic.
3- The number of bands per primer ranged from 3 to 10 bands, with an average of 7 bands per primer.
4- The highest percentage of polymorphism (%P) showed in ISSR was 66.67% generated by both UBC-840 and UBC-846 primers.
5- ISSR markers showed a polymorphism information content ranged from 0.07 to 0.23 with an average of 0.17.
6- The averaged primer resolving power generated by ISSR was 1.64 ranged from 0.8 to 2.8, while the diversity index ranged from 0.24 to 0.66 with an average of 0.46.
7- ISSR primers used in this study were able to generate only one unique band specific for landraces with high response under drought stress generated by UBC-834, while no specific bands were generated for low responsive landraces.
8- ISSR cluster analysis based on Jaccard’s coefficient of similarity was similar to SRAP based analysis in separating the highest and lowest responsive landraces from each other in two main clusters attached at similarity coefficient of 0.75.
SRAP-ISSR combined analysis
1- The combined data based dendrogram contained two main clusters similar to SRAP- and ISSR-based dendrograms.
2- Highly significant positive correlation between similarity values of SRAP and ISSR was observed (r = 0.44, P = 0.003, Mantel test).
3- There was a significant difference between the similarity values of the two molecular markers used in this study (P = 0.022, t-test).
4- Evaluating guava genotypes for drought tolerance in vitro using PEG treatment in this study was useful in determining the optimum concentration of PEG for guava in vitro screening.
5- The established method provides an excellent tool for discriminating a large number of genotypes in a short time and an inexpensive technique, which could be used with other woody plant species.
6- The molecular analysis of the highest and lowest responsive genotypes under stress using SRAP and ISSR was able to categorize tolerant and susceptible genotypes molecularly, as well as generating some unique and specific bands for each category.
7- The results of this study could be used successively in molecular breeding programs for guava improvement.