Development of PCR-based markers for thermosensitive genetic male sterility gene tms3(t) in rice (Oryza Sativa L.)

Abstract

Development of simple and reliable PCR-based markers is an important component of marker-aided selection (MAS) activities for agronomically important genes in rice breeding. In order to develop PCR-based markers for a rice thermosensitive genetic male sterility gene tms3(i), located on chromosome 6, the nucleotide sequences of four linked RAPD markers OPF18₂₆₀₀, OPAC3₆₄₀, OPB19₇₅₀ and OPM7₅₅₀ were used to design and synthesize several pairs of specific primers for PCR amplification of the genomic DNA of both the parents IR32364TGMS (sterile) and IR68 (fertile), involved in mapping this gene. For the RAPD marker OPF 18₂₆₀₀, two pairs of specific primer pair combination from different positions of the sequence resulted in generation of two codominant STS (Sequence Tagged Sites) markers. In case of markers OPAC3₆₄₀, OPB19₇₅₀ and OPAA7₅₅₀ the first two could generate dominant polymorphism, while the last one could not be successful in PCR amplification. Both the codominant STSs with primer combinations F18F/F18RM and F18FM/F18RM were found to be tightly linked to the tms3(t) gene with a genetic distance of 2.7 cM. The sizes of the different alleles in case of F18F/F18RM, F18FM/F18RM combinations were 2300 bp, 1050 bp, and 1900 bp, 1000 bp respectively. The efficiency of marker-assisted selection for this trait was estimated as 84.6%. Polymorphism survey of 12 elite rice lines, indicated that these PCR-based markers for tms3(t) can now be used in selecting TGMS plants at seedling stage in the segregating populations in environment independent of controlled temperature regime.