Emad Mohamed Hussein Ibrahim
University: Ain Shams
Date : 2017
Genetic Improvement of Chickpea ( cicer arietinuml. ) Response to Nitrogen Fixing Bacteria Via Inducation of Mutations
Nodulation mutants are one important approaches to improve nodulation for enhancing biological nitrogen fixation as a safe alternative to industrial fertilizers. In this study, developed TILLING mutant population of chickpea cultivar Ghab5 using 0.2% EMS treatment was used to look for randomly induced point mutations of 10 individual genes (EFD, ERN1, ERN2, ERN3, EIN2, RDN1, HAR1, KLV, ASTRAY, and NSP2) involved in regulation of nodule number by Illumina DNA sequencing. Total 1271 from 2500 seeds treated with 0.2 % EMS were germinated and subsequently produced seeds were used to generate M2 and M3 mutant plants. Genomic DNA of 1271 samples of M3 mutant plants were extracted, normalized in DNA concentrations and pooled to yield eight row- and 12 column-pools (for a total of 20 pools). PCR technique was conducted on a total of 20 pools with 68 TILLed fragments representative of 10 genes, only 54 amplified TILLed fragments were verified and pooled to yield eight horizontal- 12 vertical libraries (for a total of 20 libraries). Results of Illumina DNA sequencing on 20 libraries confirmed of point mutations in four different genes (one missense mutation of NSP2 gene, one nonsense mutation of EIN2 gene and two mutations in the intron regions of both RDN1and ASTRAY genes). Phenotypic data of M4 mutant plants carrying four mutants were screened, EIN2 mutant revealed superiority in supernodulating phenotype compared to the other mutants. The EIN2 M4 mutant plants were evaluated with isolates of Mesorhizobium sp. to confirm superiority in supernodulating phenotype compared with the control plants. We concluded that EIN2 mutant gene belonging to ethylene insensitive group led to supernodulating phenotype, product of EIN2 gene is a transition protein in biosynthesis of ethylene which is known as a negative regulator of nodulation processes.
Key words: Nodulation mutants– Targeted induced local lesions in genome (TILLING)– Supernodulation genes– Illumina DNA sequencing.