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Multiple loci in a continuously asexually reproducing genome such as vegetatively propagated grapevine (Vitis vinifera) can be heterozygote. The methodology to analyze heterozygous loci is manifold ranging from traditional breeding and studying segregating offspring, codominant marker analyses to whole sequence analysis. Results of heterozygosity studies on challenging loci need to be carefully confirmed to ensure accuracy and avoid misinterpretation. One of these methods is high-resolution melt (HRM) analysis in combination with sequencing and segregation analysis. We present first the adoption of HRM analyses for grapevine and its potential to confirm heterozygotic markers with low or no sequence size differences.