Salt Tolerance and Screening for Genetic Changes in Rice Mutants after Gamma Irradiation Using RAPD and Microsatellite (RAMP) Markers

Abstract

Two Vietnamese rice mutants (M2-2 and M2-4-second generation line 2 and line 4 respectively), induced by gamma irradiation were examined and compared to their parent (TT), and the Australian cultivar Amaroo (Am) for salt tolerance and genomic differences. Seedlings in tissue culture provided high yields and good quality DNA, especially with young shoots. Changes in DNA of the mutants in comparison to TT and Am were successfully assessed using RAPD-PCR (random amplified polymorphic DNA-polymerase chain reaction) and RAMP-PCR (random amplified microsatellite polymorphism-polymerase chain reaction) molecular marker techniques. RAMP-PCR was better than RAPD-PCR at disclosing genetic changes between closely related individuals, and only RAMP-PCR was able to detect polymorphic bands between the two mutants. A measure of heterozygocity and genetic differentiation, the G-statistic demonstrated that comparisons between Am and TT, M2-2 or M2-4 showed moderate genetic differentiation , but comparisons between TT and M2-2 or M2-4 showed low genetic differentiation. Percentage polymorphism between TT, M2-2, M2-4 and Am detected by RAPD-PCR were similar to those detected by RAMP-PCR (about 10%). DNA bands that were polymorphic between the two mutant lines were sequenced, two bands mapped on numerous chromosomes of rice, while two other bands mapped on one or two chromosomes, and these DNA bands on the whole coded for regulatory genes. The results showed that especially RAMP-PCR is a versatile, sensitive and cost effective method for measuring genomic differences between closely related plants.