الفهرس 
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Abstract
The present investigation was carried out at the Experimental farm and the Biotechnology laboratory of the Genetics Department, Faculty of Agriculture, Assiut University, Assuit, Egypt, during the period 2011 – 2015. The investigation aimed to study the following objective:
I. Genetic analysis of yield and yield contributing characters.
II. Genetic relationships among wheat genotypes based on agro-morphological traits.
III. Genetic variability for drought tolerance in wheat based on seedling traits and related to molecular marker:
A- Genetic variability for drought tolerance in wheat.
B- Screening wheat genotypes for DREB gene using genome specific primers.
IV. Genetic relationship among wheat genotypes based on different systems of molecular markers.
1- Seventy three wheat genotypes collected from different regions (65 bread wheat and eight durum wheat entries) were used in the present investigation. In the first part of the present investigation, the performance of these genotypes during two seasons was evaluated for fourteen characters. These characters are; heading date, plant height, number of tillers/plant, grain yield/plant, biological yield, harvest index, spike length, number of spiklets/spike, spike weight, number of grains/spike, spike density, Spikelet fertility, 1000 grain weight and awns length. For each trait the genetic, environmental and phenotypic variances, phenotypic and genotypic coefficients of variation (PCV and GCV), and broad-sense heritability (H2) are further estimated from the ANOVA mean squares. The expected genetic advance (GA) was determined at 5% and 20% intensity of selection. The simple correlation coefficients between all studied characters are also determined. In the second part, the interrelationship between the tested genotypes was studied using phylogenetic tree analysis based on morphological characters. The third part was conducted to evaluate the genetic potential of these genotypes through artificially created water stress by PEG in laboratory conditions based on easily measurable and inherited seedling traits contributing to drought tolerance. These traits included final germination percentage, shoot and root length, fresh weight and dry weight. In addition, the 73 wheat genotypes were screened for DREB gene using genome specific primers. In the fourth part of the present investigation, different systems of molecular markers (six RAPD, five ISSR, seven TRAP and eleven SSR markers) were used to study genetic relationship among the 73 wheat genotypes.
The results obtained could be summarized as follow:
I- Genetic analysis of yield and yield contributing characters.
1. Highly significant differences were found between the tested genotypes in all studied traits.
2. The genotypic component (δ2g) was greater than the environmental component (δ2e) in all studied traits.
3. In both bread and durum wheat genotypes, the phenotypic and genotypic coefficients of variation (PCV and GCV) were low for heading date and number of spikelts/spike, and moderate for plant height, biological yield, Harvest index, number of grain/spike, spike density, Spikelet fertility, 1000 grain weight.
4. The phenotypic and genotypic coefficients of variation (PCV and GCV) were high for number of tillers/plant, spike weight and awns length in bread wheat and moderate in durum wheat. Whereas grain yield/plant exhibited moderate values in bread wheat and low in durum wheat.
5. High heritability estimate associated with high genetic advance were observed for plant height and biological yield in both bread and durum wheat and for number of grains per spike in bread wheat reflecting the preponderance of additive gene action and direct selection could be made to improve these traits.
6. Heritability estimate was high and associated with moderate genetic advance were observed for harvest index in both brad and durum wheat, for days to heading, number of tillers per plant, grain yield per plant in bread wheat and for number of grains per spike in durum wheat. This indicated the presence of additive and non-additive gene action and these traits can be improved by mass selection or other breeding methods based on progeny testing.
7. High heritability estimate associated with low genetic advance were observed for spike length, number of spikelts/spike, spike weight, spikelet fertility and 1000 grain weight in both bread and durum wheat, for spike density in bread wheat, whereas for days to heading, number of tillers per plant, grain yield per plant and spike weight in durum wheat. These results indicated that these traits controlled by non-additive gene action and could be improved through hybridization and hybrid vigor.
8. Moderate heritability associated with low genetic advance were observed for awns length in both bread and durum wheat, and for spike density in durum wheat which indicated that the influence of error variance on these traits was high and suggested that indirect selection of other secondary traits may be feasible to improve them.
9. Heading date showed significant negative genotypic and phenotypic correlation with harvest index, 1000 grain weight, spike weight, awns length and grain yield per plant in bread wheat. This indicated that selection for earliness would improve grain yield in wheat.
10. Also grain yield per plant showed significant positive phenotypic and genotypic correlations with biological yield, harvest index, spike length, number of grains per spike, spike weight, percentage of spike fertility, 1000 grain weight and awns length in bread wheat. This indicated that the improvement in any of these traits might result in positive response of the grain yield in bread wheat.
11. Plant height showed significant negative correlation with grain yield per plant, awns length and spikelet fertility while it displayed positive correlation with the other characters in bread wheat.
12. In durum wheat, significant positive correlations were found between plant height and each of spike length, biological yield, 1000 grain weight and awns length as well as between grain yield per plant and number of tillers.
13. Spike length was strongly correlated with awns length and 1000 grain weight, but highly significant negative correlated with spike density in durum wheat.
II- Genetic relationships among wheat genotypes based on agro-morphological traits.
1. The Euclidean distance among all genotypes was relatively wide and ranged from 0.99 to 15.41.
2. The dendrogram distinguished 73 genotypes of wheat into eleven clusters. Cluster-1 contained the most of tetraploid wheat genotypes except G7 which separated alone in cluster-9. The other clusters contained the bread wheat genotypes, which cut off in groups according to the agro-morphological characteristics convergent.
III- Genetic variability for drought tolerance in wheat based on seedling traits and related to molecular marker:
1. Final germination percentage, shoot and root length, fresh weight and dry weight were sharply affected by drought stress in comparison to the control treatment, however, highly significant differences between the 73 tested wheat genotypes were observed.
2. Significant differences were found between the tested levels of drought stress in all traits, as well as in the interaction between PEG concentrations and the genotypes. This indicated that the genotypes performed differently from one level of drought stress to another.
3. Final germination percentage showed non-significant correlation with all traits except root dry weight under control conditions, whereas showed significant positive correlations with all characters, except shoot fresh weight under stress conditions.
4. Shoot length showed highly significant and positive correlation with root length under control and stress conditions.
5. The dendrogram based on drought susceptibility index for all traits distinguished the 73 wheat genotypes into two major clusters.Cluster-1 comprised of 20 wheat genotypes (18 bread and 2 durum wheat) which showed lowest drought stress susceptibility indices. These genotypes could be used in breeding programs to improve drought tolerance in wheat. The rest of wheat genotypes (53 genotypes) were grouped together in cluster-2 which had high drought stress susceptibility indices.
6. Screening wheat genotypes for DREB1 gene revealed that the 18 drought tolerant genotypes of bread wheat displayed the DREB1 gene in their three genomes (3A, 3B and 3D). While this gene was only amplified from the 3D-genome of three low tolerant genotypes (DSI from 0.90 to 0.93).
7. In durum wheat, the DREB1-gene was detected in both A and B genomes of the drought tolerant genotypes only (G5 and G8).
8. The DREB1-gene was not detected in all drought sensitive genotypes of durum and bread wheat.
IV- Genetic relationship among wheat genotypes based on different systems of molecular markers.
1. A total of 74 DNA fragments ranged in size from 1478 bp (OPA01) to 91 bp (UBC03) were generated from all tested genotypes using six RAPD primers.
2. Each of UBC09 and UBC03 primer amplified a maximum of 16 bands while a minimum of 6 bands were amplified by the primer OPA06. The genotype (G37) displayed the higher number of DNA fragments (52 bands) while G3 and G4 revealed the least number of amplified bands (36 bands, each).
3. Polymorphism percentage ranged from 50 % (OPA07) to 83.33% (OPA06) with an average of 68.89 %.
4. The six RAPD primers produced a total of 8 unique bands, with overall frequency of 1.33 unique bands per primer, and could be useful for genotypes identification.
5. Based on RAPD data, the UPGMA grouped the 73 wheat genotypes according to their ploidy level into two main clusters with 0.78 close-off genetic similarity. Cluster 1 including durum wheat genotypes while cluster 2 contained the bread wheat genotypes.
6. The RAPD Primer UBC09 produced a unique 290 bp fragment in drought tolerant genotypes only but not in susceptible genotypes.
7. The molecular markers data was highly significant positive correlation with morphological traits (r = 0.27: p = 0.05).
8. The five ISSR primers generated a total of 63 bands ranged in size from 170 bp (ISSR-844) to 972 bp (HB13).
9. Primer ISSR-844 amplified a maximum of 15 bands, while a minimum of 10 bands were amplified by the primer HB15. The genotypes (G29) displayed the higher number of DNA fragments (49 bands) while G55 revealed the least number of amplified bands (37 bands).
10. Polymorphism percentage ranged from 53.33 % (ISSR-844) to 78.57 % (HB12) with an average of 64.95 %.
11. Two DNA fragments at molecular size (308bp) generated by HB12 primer and one fragment (855bp) generated by ISSR-814 primer could be used as molecular markers to differentiate between bread and durum wheat genotypes.
12. The five ISSR primers produced a total of 7 unique bands, with overall frequency of 1.4 unique bands per primer, and could be useful for wheat genotype identification.
13. The dendrogram based on ISSR data grouped the 73 wheat genotypes according to their ploidy level into two main clusters with overall 0.86 genetic similarity. Cluster 1 included the durum wheat genotypes while cluster 2 included bread wheat genotypes.
14. The ISSR Primer HB13 produced DNA fragment at 597 bp in drought tolerant genotypes only, but not found in susceptible genotypes.
15. The ISSR molecular markers data showed highly significant positive correlation with morphological traits (r = 0.32: p = 0.05).
16. The Mantel test between the two Dice similarity matrices of RAPD and ISSR markers was significant (r = 0.48: p = 0.05) indicating good agreement between these two marker systems.
17. The seven TRAP markers combinations amplified a total of 86 DNA fragments from all tested genotypes and ranged in size from 987 bp (TRAP3) to 91 bp (TRAP-1).
18. Each of TRAP-2 and TRAP-5 primers amplified a maximum of 16 bands; while a minimum of 8 bands were amplified by the primer TRAP-6. The genotypes (G49) displayed the higher number of DNA fragments (69 bands) while G20, G21 and G22 revealed the least number of amplified bands (53 bands, each).
19. Polymorphism percentage ranged from 43.75% (TRAP-5) to 77.78% (TRAP-4) with an average of 65.46%.
20. DNA fragments at molecular weights 781 bp (TRAP-1) and 372 bp (TRAP-7) were present only in bread wheat genotypes, whereas absent in durum wheat genotypes. These TRAP fragments could be used as molecular markers to differentiate between bread and durum wheat genotypes.
21. Seven TRAP primers produced a total of 13 unique bands, with overall frequency of 1.86 unique bands per primer, and could be useful for genotype identification.
22. The TRAP dendrogram grouped the 73 wheat genotypes into two major clusters with genetic similarity at 0.92. While, one genotype, G49, separated in a single branch from the other genotypes, reflecting a relatively longer genetic distance from the other genotypes.
23. Two DNA fragments at molecular weights 430 bp (TRAP-4) and 271 bp (TRAP-6) were present only in drought tolerant genotypes, while it was absent in the other genotypes.
24. Highly significant positive correlation were found between TRAP markers data and morphological traits (r = 0.31: p = 0.01).
25. A total of 89 SSR alleles have been detected in 73 wheat genotypes using eleven microsatellites. The number of alleles per locus ranged from one for the SSR-11 locus to 14 for the SSR-1 locus. Their fragment size ranged from 1007bp in SSR-7 to 93 bp in SSR-10. The genotype (G29) displayed the highest number of alleles (77 alleles), while G6 (49 alleles) followed by G4 (50 alleles) revealed the least number of alleles.
26. Polymorphism percentage ranged from 50 % (SSR-1, SSR-2 and SSR-5) to 100% (SSR-10 and SSR-11) with an average 68.24%.
27. Eleven alleles at molecular size [219 bp and 188 bp (SSR-1), 239 bp (SSR-3), 635 bp and 503 bp (SSR-4), 358 bp and 331 bp (SSR-8), 250 bp, 226 bp and 210 bp (SSR-9) and 532 bp (SSR-11)] were present only in bread wheat genotypes, while one allele at molecular size 312 bp (SSR-6) was unique to durum wheat genotypes. These alleles could be used to distinguish the durum wheat from the bread wheat genotypes.
28. The SSR primers produced a total of 9 unique bands could be used for identification and discrimination.
29. The dendrogram based on SSR data grouped the 73 wheat genotypes according to their ploidy levels into two main clusters within a close-off 0.79 GS. Cluster 1 included the durum wheat genotypes, while cluster 2 contained all bread wheat genotypes.
30. Three DNA fragments at 529 bp (SSR-5), 554 bp (SSR-6) and 412 bp (SSR-8) were detected only in drought tolerant genotypes, but not in susceptible genotypes. These fragments could be used as specific markers for drought tolerance in wheat.
31. The SSR markers data showed highly significant positive correlation with morphological traits (r = 0.42: p = 0.01).
32. The combined data of RAPD, ISSR, TRAP and SSR markers revealed that a total of 312 DNA fragments were amplified by the 29 primers from all 73 genotypes with an average 10.76 bands/primers. Out of these fragments 201 (64.42%) showed polymorphism and 111 (35.58%) bands were common, monomorphic, in all genotypes.
33. The genotype (G49) displayed the highest number of DNA fragments (239 bands) followed by G29 and G28 (236 and 233, respectively), while the genotype (G6) revealed the least number of bands (185 bands).
34. The Mantel test values between Dice similarity matrices of SSR with RAPD, SSR with ISSR, SSR with TRAP and SSR with RAPD+ISSR+ TRAP markers were significant (r = 0.75, r = 0.0.59, r = 0.68 and r= 0.84, respectively), which indicated good agreement between RAPD, ISSR, TRAP and SSR marker systems.
35. These results indicated that the dendrogram, based on different systems of molecular markers (i.e. RAPD, ISSR, TRAP and SSR markers), was able to classify wheat genotypes according to their origin.
36. The molecular markers data revealed highly significant positive correlation with morphological traits (r = 0.43: p = 0.01). The significant correlation indicate that these independent sets of data likely reflect the same pattern of genetic diversity and validate the use of these data to calculate the different diversity statistics for morphological traits in the wheat genotypes.