Determination of Genetic Purity of Hybrid Seed in Watermelon (Citrullus lanatus) and Tomato (Lycopersicon esculentum) Using Random Amplified Polymorphic DNA(RAPD).

Abstract

To determine the genetic purity of hybrid seeds, RAPD was employed in order to discriminate parents and their hybrids using a single primer for each reaction. Fifty-nine oligonucleotides were screened as primers for polymerase chain reaction (PCR) in inbred parental lines (Ha,Hb) currently used for commercial seed production in watermelon. Only 3% of the totally amplified DNA showed polymorphism, suggesting that there was a close relationship between the parents. The PCR using nine primers resulted in polymorphism. One of the primers, RA12-12(5'-ACCACCTGGCTC-3'), generated a product specific to the F-1 and male parent(Hb), and enabled to discriminate the female (Ha) from the hybrid. In tomato, twelve primers were screened to discriminate two inbred lines, 'Morioka No.20' (M) and 'Ottawa 48' (O), and four showed polymorphism. One out of four enabled to distinguish the female line (M) from the F-1 (M times O). Thirty primers were screened to segregate another pair of parents, O and 'Veeking' (V), and five generated polymorphism. One of them enabled to discriminate the F-1 from O. Four to seven percent of the total PCR products showed polymorphism, suggesting that there were close relationships among the parental lines. Using these primers, the conditions required for PCR and a method for DNA preparation were also studied. When 1 to 100 ng of template DNA was used per reaction mixture, the electrophoretic pattern of the PCR product remained constant. When DNA prepared with a simple method (EDWARDs et al. 1991) was used as template, the results were essentially the same as those obtained with the purified DNA. Determination of the genetic purity of F-1 hybrid seeds by RAPD was discussed along with the close relationship between the parents of the vegetable crops, where inbred depression is not conspicuous.