Advisor : Moharil, Dr. M. P.Authors : SHARMA, PALAK.2022-02-262022-02-262022-07-20Citation : SHARMA, PALAK. (2021). Differential gene expression profiling during Fusarium wilting in chickpea using cDNA SCoT markers. Biotechnology Centre, Department of Agricultural Botany. Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. M. Sc. 2021. xiv, 107p. Print. (Unpublished)https://krishikosh.egranth.ac.in/handle/1/5810182960Description : Gene expression profiling measures which genes are being expressed in a cell at any given moment. This method can measure thousands of genes at a time; some experiments can measure the entire genome at once. Gene expression profiling measures mRNA levels, showing the pattern of genes expressed by a cell at the transcription level. This often means measuring relative mRNA amounts in two or more experimental conditions, then assessing which conditions resulted in specific genes being expressed.Abstract : Chickpea (Cicer arietinum L.) is an annual, autonomous legume cultivated over 30 different countries having genome size of 740 Mbp and has a place within family Leguminoseae. India is the fundamental and prime producer of the chickpea contributing 65% in total world chickpea production and 75% of area coverage in the world. Various abiotic and biotic stresses are responsible for the loss in chickpea productivity. Fusarium wilt, caused by Fusarium oxysporum f. species ciceris is an economically important disease of chickpea. The pathogen can survive in soil for more than six years even in the absence of chickpea and causes 10%-15% yield losses annually. Keeping in view of importance of this crop and potential threat of Fusarium wilt in chickpea growing areas and to address the need for genomic tools in chickpea for Fusarium wilt resistance, the present investigation entitled “Differential Gene Expression Profiling During Fusarium Wilting in Chickpea Using cDNA SCoT Markers” was undertaken to determine differential gene expression analysis using cDNA-SCoT profiling to identify differentially expressed fragments (DEF’s) in resistant and susceptible cultivars. This study focused on which genes are responsible for Fusarium wilt resistance in the chickpea genotypes. This programme was carried out during 2019-21 at Biotechnology Centre, Department of Agricultural Botany, Dr. Panjabrao Deshmukh Krishi Vidyapeeth, Akola. Pathogenicity test of Fusarium oxysporum f.sp.ciceri by root dip inoculation method was performed which showed 84 percent wilting in susceptible cultivar at 25th days after inoculation whereas, in resistant cultivar few plants with wilting symptoms were observed. Characterization of resistant and susceptible genotypes of chickpea using quantified cDNA of both plant samples was profiled using 13 SCoT primers, all primers showed reproducibility. About 77 amplicons were generated amongst that 63 were polymorphic and 14 were monomorphic. The polymorphic amplicons were re-amplified (nested PCR) and sequenced using Applied Biosystem by Sanger sequencing method. The DEF’s were further analyzed using computational tools viz., BLAST homology, string protein interaction. Similarity search showed that both the differentially expressed fragment showed maximum homology by i.e 84.21% and 91.43% with Cicer arietinum cysteine rich PAK1 inhibitor like mRNA (LOC101498699) which act as a defensin like peptide. The P21 activated kinase 1 (PAK1) inhibitory compound having antifungal activity. Plant cysteine rich peptides were able to inhibit pathogenic growth and helps the legume formation in plants. String protein interaction studies revealed that the target protein interacts with the regulatory proteins like Receptor-like protein 12; Leucine-rich repeat receptor protein kinase EXS-like which are Leucine Rich Receptor Like Kinase contributes to Fusarium resistance in cereals. Cicer arietinum cystine rich receptor-like protein kinase (XP_004500476.1) interact with many other proteins which suggest the suitability of using this gene as a candidate gene in development of transgenic plants resistance against Fusarium wilt. The cDNA-SCoT technique is becoming increasingly important in the study of differentially expressed genes, the discovery of novel genes, and the exploration of molecular pathways. It should be made available to future researchers as an alternative to cDNA-AFLP, cDNA-SRAP, and other comparable technologies for multifunctional plant gene differential expression applications and studies. The information gained from identifying differentially expressed DEFs in tissues of infected and uninfected plants could be used to predict gene(s) involved in Fusarium wilt resistance in chickpea genotypes, for further, more detailed studies.EnglishThesis