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Abstract This study was carried out in the laboratories of the Genetics Dept., Fac. of Agric., Ain Shams Univ. during the period from 2008 to 2014. Biodiversity refers to variation within the living world, while genetic diversity represents the heritable variation within and between populations of organisms. Transposable elements (TEs) are DNA elements that can transpose from one place in DNA to another. There are two main classes of TEs; DNA transposons; which act through a DNA intermediate, move from site-to-site through a cut-and-paste mechanism and multiply by using the host cell’s replication machinery, and retroelements (retrotransposons); which act through an RNA intermediate, move from site-to-site through a copy-and-paste mechanism. Retrotransposons proved to be useful for molecular genetic studies and play an important role in genome evolution. five molecular genetic techniques (RAPD, ISSRs, IRAP, REMAP and RRAP) are used to study the role of retrotransposons in genomic diversity in different eukaryotic organisms such as loweringeukaryotic single-cell organisms (yeast) and higher-flowering-plants (barley). The ojectives were:- 1. To study the effectiveness of the retrotransposon-based techniques (IRAP & REMAP) to clarify genomic diversity. 2. To evaluate the efficiency of new developed RRAP technique in detection of genetic diversity and in comparison with other retrotransposon-based techniques (IRAP & REMAP). 3. To compare the retrotransposon-based techniques (IRAP & SUMMARY Marwa M. Shehata, M.Sc., 2014 102 REMAP) with the traditional molecular techniques such as RAPD & ISSRs-PCR. Five yeast strains (UQM 49, NRRL Y-17008, LBC 1208, LBC 254 and ATCC 58523) and six barley cultivars (Giza 123, Giza 126, Giza 129, Giza 130, Giza 131 and Giza 2000) were used as DNA sources to assess the genetic diversity among yeast strains and barley cultivars. Two retrotransposon-based primers (ScM1 and ScM2) were designed using ten Ty1-LTR-retrotransposon sequences (YBLWTy1- 1, YBRWTy1-2, YDRCTy1-1, YDRCTy1-2, YDRWTy1-4, YDRWTy1-5, YERCTy1-1, YERCTy1-2, YGRCTy1-2 and YGRCTy1-3) and three freeware (Clustal X, CLC Free Workbench and PerlPrimer) and one commercial software (Lasergene7-PrimerSelect). To study the genetic diversity the different genotypes using non-retrotransposon-based techniques, ten RAPD and nine ISSRs primers were used. On the other hand, using retrotransposon-based techniques (IRAP, REMAP and RRAP), three and five IRAP primers and their combinations were used for study the genetic diversity of yeast strains and barley cultivars, respectively. Eleven combinations of IRAP and ISSRs primers were used to study the genetic diversity for yeast strains via REMAP technique. And 16 REMAP combinations were used for barley cultivars. RRAP technique was performed using combinations of IRAP and RAPD primers. Eleven and 13 combinations were used to study the genetic diversity of yeast strains and barley cultivars via RRAP technique, respectively. SUMMARY Marwa M. Shehata, M.Sc., 2014 103 The following results were obtained: 1. In yeast strains, RAPD primers showed highly polymorphism percentages where all primers displayed more than 75% polymorphism percentages. Primers polymorphism percentages ranged from 77.78 (D15) to 100% with three different primers (A15, B08 & O14). Mean of band frequency ranged from 26.25% to 65%. RAPD primers differed in their amplified loci (band number) from just eight loci in B15 primer to 31 different bands in O10 RAPD primer. 2. ISSRs primers were highly polymorphic where about 67% of the used ISSRs primers displayed more than 75% polymorphism percentages. Primers polymorphism percentages ranged from 46.15 (primer 814A) to 100% with only one primer (844B). The primer 814A the highest mean of band frequency (76.92%). Also, the 100% polymorphic primer 844B was the lowest primer in band frequency (38.00%). ISSRs primers amplified similar loci numbers on yeast chromosomes where most of the ISSRs primers (seven of nine ISSRs primers) displayed from ten (844B) to 16 (17899B) bands. Three different primers (17898B, HB12 and HB14) showed the same number of bands (14 bands). Only two primers amplified more loci generating more than 16 bands; HB13 (20 bands) and HB15 (24 bands). 3. IRAP primers showed moderately polymorphism where the two primers displayed less than 75% polymorphism percentages. Only the combination between the two primers revealed more than 75% polymorphism (81.82%) showing a synergism effect with 58.18% mean of band frequency. On the other hand an antagonism effect appeared in the band SUMMARY Marwa M. Shehata, M.Sc., 2014 104 number. ScM1 alone amplified 13 loci and ScM2 represented 19 bands. While the combination of them together displayed bands less than any of them alone (11 bands only). 4. REMAP primers showed moderately to highly polymorphism percentages where four of 11 combinations displayed more than 75% polymorphism percentages. Combinations polymorphism percentages ranged from 33.33% (ScM2+17898B) to 88.46% (ScM2+HB14). The lowest mean of band frequency was 60.00% which observed in the combination between ScM1 and HB12 primers. The number of bands in the REMAP technique was relatively high. The lowest number of bands was 16 bands of the combination ScM1+17899B, while the bands number for the combination ScM1+HB13 showed the highest value (33 bands). 5. RRAP primers showed moderately to highly polymorphism percentages where four of 11 combinations displayed more than 75% polymorphism percentages. Combinations polymorphism percentages ranged from 31.25% (ScM1+B08) to 100% (ScM1+A20) with a mean of band frequency 80.00% and 43.81%, respectively. RRAP combinations showed similar number of bands. Among 11 combinations six of them were in the range between 15 bands (ScM1+A15 and ScM2+B17) to 19 bands (ScM1+O14). The highest bands number was observed in the combination ScM2+O14. 6. In barley cultivars, RAPD primers showed variable polymorphism percentages. B10 displayed the lowest polymorphism percentage (16.67%) and the highest mean SUMMARY Marwa M. Shehata, M.Sc., 2014 105 of band frequency (90.25%). While, A15 showed the highest polymorphism (100%) and the lowest band frequency (45.33%). About 40% of the primers were highly polymorphic which displayed more than 75% polymorphism. Also, RAPD primers were different in their amplified loci or generated bands. About 60% of the primers showed less than 20 bands per primer. Primers A20 and D15 displayed the highest band number or loci number per primer (26). 7. ISSRs primers showed moderatly polymorphism. All primers displayed equal or less than 75% polymorphism. One primer represented 0% polymorphism (HB14). The mean of band frequency per primer ranged from 58.35% to 100.00%. All ISSRs primers showed more than 50% mean of band frequency. Except 17899B primer, all ISSRs primers were similar in their generated bands, where the number of bands through all ISSRs primers except 17899B showed from six to 14 bands per primer. Only the excepted primer 17899B represented 22 bands. 8. Both 5’LTR and sukkula primers displayed highly polymorphism (89.47 and 85%, respectively). Whereas, Wltr2105 primer showed low polymorphism percentage 12.5%. The combinations between primers showed either low or moderately polymorphism. The mean of band frequency ranged from 47.36% to 97.91%. The number of bands was similar among the IRAP primers and their combinations which were from 19 to 25 bands. The only exception was for the primer Wltr2105 which displayed only eight bands. SUMMARY Marwa M. Shehata, M.Sc., 2014 106 9. REMAP technique revealed moderate polymorphism. The polymorphism percentage per primers-combination ranged from 11.11% (5’LTR+HB13) to 61.90% (Wltr2105+HB13). All combinations except two of them displayed less than 50% polymorphism. On the other hand, only three of 16 primer-combinations showed less than 25% polymorphism. The mean of band frequency ranged from 72.23% (Wltr2105+844B) to 96.30% (5’LTR+HB13). REMAP combinations showed similar numbers of bands. Three of them represented the same number of bands (14 bands). Two combinations showed 21 bands per combination, 16 bands per combinations are reported in two different combinations and another two combinations revealed the same number of bands again (nine bands). 10. RRAP technique revealed low to moderately polymorphism. Most primer-combinations showed polymorphism percentages between 25 and 50%. Only two combinations displayed more than 50% polymorphism. And four combinations were lower than 25%. The mean of band frequency ranged from 69.69% (5’LTR +O10) to 97.22% (5’LTR+O14). Number of bands among the RRAP technique was almost similar. Three combinations showed 19 bands. Two other combinations revealed 17 bands. The range of bands were from 11 (5’LTR+O10) to 27 (5’LTR+B08) bands. 11. Wide range of temperature degrees was used to detect the optimum annealing temperature for REMAP and RRAP experiments in barley cultivars. It was appeared that 50oC is the optimum temperature for all REMAP and RRAP combinations. Different banding patterns were observed in SUMMARY Marwa M. Shehata, M.Sc., 2014 107 REMAP and RRAP combinations using the same IRAP primer at the annealing temperature of 50oC. 12. The comparison among the five techniques based on their ability to produce polymorphism between the studied DNA samples showed that: a. RAPD technique represented the highest polymorphism percentages per primer among the other four techniques. b. ISSRs primers represented polymorphism percentages more than IRAP primers in yeast but IRAP primers were more polymorphic than those of ISSRs in barley. c. Both REMAP and RRAP combinations showed similar results either in yeast or barley. d. On the other hand, yeast strains displayed polymorphism values more than those of barley cultivars. e. Retrotransposon-based techniques showed more number of bands more than those of nonretrotransposon- based techniques. REMAP and RRAP produced the highest band numbers (41 and 40, respectively). While, RAPD, IRAP and ISSRs displayed 37, 33 and 27 bands, respectively. f. Both yeast strains and barley cultivars showed specific ”unique” bands. The five yeast strains revealed 178 unique bands, most of them were observed in the strain ATCC-58523 (S5). Seventy four cultivar-specific bands were observed in the six barley cultivars. Giza-123 cultivar showed the SUMMARY Marwa M. Shehata, M.Sc., 2014 108 highest number of these bands. Among the five techniques, RAPD markers represented about 40 and 54% of the unique bands in yeast and barley genotypes, respectively. 13. Retrotransposons based molecular markers have many advantages than using random primers as RAPD and ISSRs techniques make them a useful approach as molecular markers such as repeatability and reproducibility. The 5’LTR primer with the six barley cultivars were used together in different times. However, the obtained patterns were similar, so the retrotransposons-based techniques were an important technique in detecting the genetic diversity and gave a higher repeatability. As a conclusion, the ideal molecular marker technique should have specific criteria. As yet, no molecular marker has been known to have all of the aspects together. Retrotransposon-based methods can be generalized, furthermore, to other transposable element systems and to other organisms due to their positive features as molecular markers. |