Development of PCR Primers Diagnostic for Cytoplasmic Male Sterile and Fertile Maintainer Lines of Mestizo Hybrid Rice

Abstract

Ten DNA primers were designed for the PCR amplification of specific regions of mDNA atp6 gene of Mestizo hybrid rice. The design was based on known sequences with accession numbers E104444 and X52162 of cytoplasmic male sterile (CMS) lines and fertile lines in GenBank found in the National Center for Biotechnology Information (NCBI) database. The objective of the development of these particular DNA primers was to distinguish the CMS line (IR58025A) from the maintainer line (IR58025B) and from other rice lines. The optimization of the PCR conditions using the DNA primers was done. The conditions optimized were annealing temperature, MgCl2 concentration and DNA template concentration. Among the ten primers constructed, a6P5 and MdFI were found to amplify the mDNA atp6 gene of rice and can be potential markers for discriminating CMS line from maintainer line. The optimum PCR conditions for these primers are: 52˚C annealing temperature, 1.00nM MgCl2 and 250 ng template DNA concentration. The a6P5 primers are characterized by a 300bp product size, with its left primer having 22 base pairs and 40.91 GC% and the right primer with 19 base pairs and 52.63 GC%. The MITdelF1 primers are characterized by a 502bp product size, with the left primer having 24 base pairs and the right primer with 20 base pairs and GC% of 37.5 and 50 respectively.