Development of a STS marker assay for detecting loss of heterozygosity in rice hybrids

Abstract

The RAPD (random amplified polymorphic DNA) markers OPE15750 and OPE15300 were affected by loss of heterozygosity (LOH) in rice hybrids AMR × 'M202' and AMR × 'L202'. The markers were mapped to the same locus at or near the centromere of rice chromosome 2. The two RAPD products were cloned, sequenced, and found to have lengths of 734 bp and 297 bp, respectively. The 297-bp sequence shares a 98% homology with one end of the 734-bp sequence, accounting for the cross-hybridization previously observed between the two clones. Based on the DNA sequence of the 734-bp fragment, a pair of STS (sequence-tagged site) primers was designed and tested. Rice plants homozygous for either OPE15734 or OPE15297 all produced PCR fragments of the same length, 482 bp. However, the two PCR products were discernible by digestion with the restriction enzyme XbaI prior to gel electrophoresis. The STS product from plants homozygous for OPE15734 was cut into two fragments of 239 and 240 bp, which appeared as one single band in an agarose gel; whereas the STS product from plants homozygous for OPE15297 was not cut by XbaI and was characterized by a 482-bp band in the agarose gel. These STS primers were tested in rice hybrids and F2 progenies derived from the hybrids of AMR × 'M202' and AMR × 'L202'. Homozygosity for OPE15297 was confirmed for all F2 panicle rows derived from AMR × 'M202'. In contrast, F2 panicle rows of AMR × 'L202' exhibited two different segregation patterns (genotypes), i.e., either uniformly homozygous for the 240-bp marker (OPE15734/OPE15734) or segregating for the 482- and 240-bp markers (OPE15734/OPE15297). This STS-marker system provides a robust assay for detecting the occurrence of LOH in rice hybrid progenies.Key words: DNA sequence, homology, PCR, RAPD.