الفهرس 
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Abstract
SUMMARY
This investigation was carried out in the Genetics
Department, Faculty of Agriculture, Ain Shams University and the
Gene Expression and Regulation Technology laboratory (GERT),
Agricultural Genetic Engineering Research Institute (AGERI),
Agriculture Research Center (ARC) Agriculture Research Center
(ARC) and International Center for Agricultural Research in the Dry
Areas (ICARDA) during the period from 2013 - 2017.
The objectives of this study were to improve nodulation of
chickpea cultivar ”Ghab 5” using induction of mutation for
enhancing nitrogen fixation. TILLING technique was used to create
random induced point mutations using EMS treatment and
identification of randomly induced point mutations in some
individual genes involved in regulation of nodule number by
Illumina DNA sequencing.
The major findings of this study were as follows:
1- The ethyl methane sulfonate (EMS) was used as a chemical
mutagen for development of TLLING population in chickpea
cultivar ”Ghab 5”.
2- A standard concentration of 0.2% (EMS) was determined to
start an experiment with 2500 seeds approximately.
3- Only 1370 M1 seeds were germinated in field from a totally
2500 seed treated with 0.2 % EMS.
4- Only 1271 M1 mutant plants gave seeds from 1370 seeds
germinated.
5- The seeds of 1271 M1 mutant plants were harvested
separately to generate M2 and M3 mutant plants.
SUMMARY
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Emad M. H. Ibrahim (2017), Ph.D. Fac. Agric., Ain Shams Univ.
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6- Genomic DNA of the 1271 samples from M3 mutant plants
were extracted and normalized with spectrophotometer.
7- Normalized DNA of 1271 samples were arrayed in 14 plates
as one 96-well per “destiny plate”, 14 individuals per well,
then the row and column samples are pooled to yield 8 rowand
12 column-pools (for a total of 20 pools).
8- Ten nodulation genes involve in regulation of nodule number
in chickpea were identified based on the results of homology
BLAST with source genes in models legume Medicago and
Lotus.
9- Software of program primer 3 was used to design 68
primers represented 68 TILLed fragments of 10 nodulation
genes of chickpea.
10- The total 68 TILLed fragments included 8 of gene A, 2 of
gene B, 7 of gene C, 5 of gene D, 12 of gene E, 8 of gene F,
6 of gene G, 6 of gene H, 10 of gene I, and 4 of gene J.
11- The total 68 TILLed fragments were amplified from 20 pools
separately; only 54 TILLed fragments were verified on 1.2 %
agarose gel electrophoresis.
12- PCR products of the 54 verified TILLed fragments were
arrayed, so that the rows and columns PCR product are
pooled to yield 8 row- and 12 column-libraries (for a total of
20 libraries).
13- MiSeq Illumina DNA sequencing was used to read the 54
amplified TILLed fragments in each Library compared to the
control, only 35 readable TILLed fragments were obtained.
14- Five different induced SNPs mutations were obtained; two
SNPs mutations in J2 TILLed fragments, one SNP mutation
SUMMARY
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Emad M. H. Ibrahim (2017), Ph.D. Fac. Agric., Ain Shams Univ.
81
in E5 TILLed fragments, one SNP mutation in F3 TILLed
fragments and one SNP mutation in I6 TILLed fragments.
15- Each induced SNP mutation was confirmed twice, once in
the horizontal library and once in the vertical library. The
reading depth of each induced SNP mutation was
determined.
16- The analysis of DNA star program results revealed the
presence of missense mutation in NSP2 gene, nonsense
mutation in EIN2 and on SNP mutation in the intron regions
in both of RDN1 and ASTRAY genes.
17- Based on the results of Illumina DNA sequencing. Only 53
plots of M4 mutant plants that carried four mutants (NSP2,
EIN2, RDN1, and ASTRAY) were screened, three plants
were taken randomly from each plot to estimate the average
of three phenotypic traits (wet weight, dry weight and the
number of nodes).
18- During the screening of the M4 mutant plants of plot No. 548
that carry the induced point mutation; stop codon in
ethylene-insensitive protein2 gene (EIN2) revealed
supernodulating phenotype which was better than the other.
19- The M4 mutant plants of plot no. 548 that carried the
induced point mutation stop codon in ethylene-insensitive
protein2 of gene (EIN2) were evaluated with four isolates of
Mesorhizobium sp. to confirm the superiority of isolate no.1
in supernodulating phenotype compared to the control
plants.
In conclusion, M4 mutant plant of plot no. 548, in this study,
carried stop codon mutation in EIN2 gene involved in regulation of
nodules number. These plants revealed an increase in the number
SUMMARY
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Emad M. H. Ibrahim (2017), Ph.D. Fac. Agric., Ain Shams Univ.
82
of root nodes than other plots, and after evaluating their response
under different bacterial isolates of Mesorhizobium Sp. they
confirmed their superiority in the number of root nodes compared
to the control plants. In the future, these mutant plants can be
exploited in breeding programs of chickpeas to increase the
formation of root nodes for biological nitrogen fixation