Sarmah, Bidyut KumarKakati, Indrani2019-06-202019-06-202017-01http://krishikosh.egranth.ac.in/handle/1/5810109371Bruchid beetles, (Callosobruchus spp.,) are a devastating stored grain pest of black gram. The interaction between bruchid and black gram genotype has not yet been demonstrated. In the present investigation, an attempt has been made to understand the molecular basis of resistance in a mild tolerant genotype (IC8219) of black gram through two molecular techniques, Suppression Subtractive Hybridization (SSH) library preparation and RNA Sequencing (RNASeq). This is the first report on elucidation of the molecular basis of defense during black gram-bruchid interaction. The changes related to generation of ROS and expression of defense related genes in a mild tolerant (IC8219 genotype) of black gram was studied after releasing bruchids. After 7 days of releasing bruchids, RNA from developing seeds of the pods oviposited by bruchids were collected. The generation of ROS was detected by using 3, 3’ diaminobenzidine (DAB) staining on the pods oviposited by bruchids. For suppression subtractive hybridization (SSH) the pods oviposited by bruchids were used as tester population, while RNA from seeds of control plants were used as driver population. A forward subtractive cDNA library was prepared from tester and driver population and subtracted cDNAs were cloned and transformed in JM109 competent cells. In all, 277 clones in an EST library were sequenced and analyzed. High quality ESTs (244) were submitted to the NCBI database (Acc. No. JZ917400-JZ917463). Based on CAP3 assembly, 134 genes were computationally annotated. The majority of the ESTs belonged to ‘Biological Process’ of gene ontology category. A total of 18 defense related genes were identified and were subjected to quantitative PCR analysis (qPCR). Of these 12 genes showed up-regulation in developing seeds. Few major defense genes like defensin, pathogenesis related protein (PR), lipoxygenase (LOX) showed high expression in the oviposited plants when compared with the non-oviposited plants. In order to obtain a greater representation of defense genes, de novo transcriptome assembly of RNA extracted from the pods oviposited by bruchids was also performed. RNASeq analysis revealed a total of 12,081 differentially expressed genes (DEGs) out of which 258 were up-regulated and 310 were down-regulated during black gram- bruchid interaction. In all, 20 defense related genes were identified of which 9 were represented both in the SSH library and RNASeq data. This is the first report on defense related gene expression study in developing seeds of black gram during upon egg laying by bruchid beetles.ennullThesis