Dr. Subhash N.RATHOD DIVYARAJSINH VIRENDRASINH2018-01-242018-01-242016http://krishikosh.egranth.ac.in/handle/1/5810039830Experiments were conducted to screen and validate suitable male specific markers in the identification of sex in papaya (cv. Madhubindu). From the reported markers W11, T12, Napf and CPSM 90 showed detectable results in sex determination and was used for further experimentation with routinely used PCR method as well as loop mediated isothermal amplification (LAMP). Using screened markers unknown samples were identified with 100% efficiency of the marker used in predicting the sex type. At the time of flowering all the predicted males as well as female were confirmed visually. Moreover, after the detection with PCR, identification of papaya DNA samples were carried out with turbidity method, fluorescence method (Calcein, SYBR green, HNB, EtBr dyes). Using four primers i.e. W11, T12, Napf and CPSM90, LAMP reaction was performed initially with confirmed male and female DNA samples. Among the various dyes used, detection with calcein dye was most revealing and intense fluorescence colour was obtained. Among various primers used T12 primer was found most prominent. With the use of T12 primer and calcein detection system, various experiments were conducted for optimization of reaction time, temperature, starting quantity of DNA and concentration of enzyme. Thus in this study 4 ii male specific markers were developed for sex identification using both PCR and loop mediated isothermal amplification (LAMP). LAMP reaction carried out by T12 primer using calcein dye showed promising results in terms of rapid reaction time (< 1h), isothermal condition (55°c), a high efficiency (20 ng of DNA required in reaction mixture) and concentration of enzyme (8 units of the enzyme).enagriculture, biotechnologyIDENTIFICATIONThesis