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Book of Abstracts First Legume Society Conference 2013: A Legume Odyssey Novi Sad, Serbia, 9-11 May 2013
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Application of HRM analysis for genetic mapping of STS markers in Lupinus
angustifolius L.
Katarzyna Kamel, Magdalena Kroc, Wojciech Święcicki
Institute of Plant Genetics, Polish Academy of Sciences, Poznań, Poland
High resolution melting analysis (HRM) is a powerful technique used for genotyping and
mutation scanning. This method takes advantage of special saturation dyes properties that
fluoresce only in the presence of double stranded DNA. After the PCR, in the presence of the
dsDNA-binding fluorescent dye, amplicons are denaturated in high temperature and the
fluorescence fades away. Fluorescence changes during the analysis can be applied for the
detection of SNP polymorphism, small deletions or insertions using melting curves as, various
genetic sequences melt at different temperature. The usability of the HRM assay has been
confirmed for genotyping of various plant and animal species. The main goal of this study is to
evaluate the effectiveness of HRM analysis for sequence tagged site (STS) markers genotyping in
the narrow-leafed lupin (Lupinus angustifolius L.). The RIL mapping population 83A:476 x P27255
was tested for several legume anchor markers (Leg markers). HRM assays were conducted on
LightCycler 480 and LightCycler 480 High Resolution Melting Master (Roche). STS markers,
which produced specific, 200-1000 bp–long products were sequenced. Markers containing SNPs
with product length greater than 300 bp were converted into smaller markers by designing new
primers sets spanning fragment of the entire sequence with SNP. Next, markers were tested with
the HRM technique in the parental lines of a mapping population. If the parental genotypes were
easy to distinguish, markers were then analyzed in the remaining lines of mapping population and
assigned to linkage groups of the narrow-leafed lupin.
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