الفهرس 
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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 &
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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.
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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
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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
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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.
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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
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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
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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.