Identificaiton and relative expression of Bacterial leaf blight resistance genes (xa13 and Xa21) in Indian basmati rice (Oryza Sativa L.)

Abstract

Thesis Title: "Identification and relative expression of bacterial leaf blight resistance genes (xal3 and Xa21) in Indian basmati rice (01yza satil1a L.)" The present study entitled "Identification and relative expression of bacterial leaf blight resistance genes (xa13 and Xa21) in Indian basmati rice (01yza sativa L.)" was carried out using thirty varieties for screening of bacterial blight resistance genotype and for expression analysis. According to first objective we were planted 100-I20 plants of each rice genotype in plots with three replications. Samples were collected and stored at -80° C. Genomic DNA was isolated from thirty rice samples by CT AB method and purified the genomic DNA. PCR was done by using gene specific primers (Xa4, xa13, and Xa21) . And PCR products was electrophoresed 2% agarose gel. The analysis of band with different size was done in gel documentation system. After analysis of banding pattern, Fifteen out of thirty rice genotypes showed the appropriate amplification for BLB resistant fragments of Xa4 gene, Existence of xa13 gene in rice germplasm was amplified through SNP marker (RG 136) and showed the I kb positive fragment in twenty one varieties out of thirty and Examination of Xa21 gene carrying rice gem1plasm by STS marker pTA248 showed the BLB resistant (R) fragment as 1 000 bp. Seven out of Thirty rice genotype showed the appropriate amplification for BLB resistant fragments of Xa21 gene. After that I was selected two isogenic lines viz. Improved Pusa Basmati-1 and Pusa Basmati-! for expression analysis. TheXanthomonas 01yzae pv. 01yzae (Xoo) obtained from IARI Delhi, was used in the present study. The culture of strain of BLB (Xoo) pathogen was multiplied and maintained on Wakki Motto media incubated at 28 °C. An average of five leaves per plant was inoculated with bacterial suspension of BLB pathogen using scissor at two different stages i.e. tillering and adult stage. Negative control was inoculated with sterile water. The samples were taken from Pusa Basmati-! and Improved Pusa Basmati-1 with different treatment (mock treatment and BLB treatment) at different time interval(O hour, 6 hour, 12 hour, 24 hour, 36 hour, 48 hour, 60 hour and 72 hour). RNA isolation and purification was carried out by TRizol method then quantificate the total RNA and prepared eDNA by using M MUL V RT PCR Kit. Set the real time PCR and conduct experiment by placing samples in 96 well plates using master mix with Syber green dye. The Ct value were used to calculate the 2- MCt of each samples. This nonnalized expression showed, the genes (xal3, Xa21) was higher expressed than control and negative control at tillering as well as adult stage. The 2-l1llct value of Improved Pusa Basmati-1 (BLB treated) form xa13 and Xa21 allele at tillering as well as adult stage explaining that the allele Xa21 is highly expressed than the xa13 allele at both tillering and adult plant stage but at adult stage the xa21 downregulated during 72 hour in relation to other time periods.