Use of Haploid Mixtures and Heteroduplex Analysis Enhance Polymorphisms Revealed by Denaturing Gradient Gel Electrophoresis

Abstract

PCR-based codominant genetic markers were developed by using primer sequences designed from cDNA clones of loblolly pine (Pinus taeda L.). Such markers offer certain advantages relative to simple-sequence repeat (SSR), also known as short-tandem repeat (STR) markers, and include the ability to quantify and map DNA polymorphisms in expressed genes. However, detecting these DNA polymorphisms is more problematic because many DNA polymorphisms in genes involve base substitutions rather than insertions or deletions. Denaturing gradient gel electrophoresis (DGGE) is a sensitive and efficient method for detecting sequence differences among PCR fragments. This paper demonstrates the application of DGGE to genetically map expressed genes in loblolly pine. Also, heteroduplex DNA fragments, formed during the amplification of DNA from heterozygotes and from mixes of haploid DNAs from megagametophytes, enhanced and strengthened genetic interpretations and genotypic classifications.