الفهرس | Only 14 pages are availabe for public view |
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 ________________________________________________________ Emad M. H. Ibrahim (2017), Ph.D. Fac. Agric., Ain Shams Univ. 80 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 ________________________________________________________ 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 ________________________________________________________ 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 |