Universität zu Köln

Kuster, C. J. and Von Elert, E. (2012). High-resolution melting analysis: a genotyping tool for population studies on Daphnia. Mol. Ecol. Resour., 12 (6). S. 1048 - 1058. HOBOKEN: WILEY-BLACKWELL. ISSN 1755-098X

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Abstract

Determining genetic variation at the DNA level within and between natural populations is important for understanding the role of natural selection on phenotypic traits, but many techniques of screening for genetic variation are either cost intensive, not sensitive enough or too labour- and time-consuming. Here, we demonstrate high-resolution melting analysis (HRMA) as a cost-effective and powerful tool for screening variable target genes in natural populations. HRMA is based on monitoring the melting of PCR amplicons. Owing to saturating concentrations of a dye that binds at high concentrations to double-stranded DNA, it is possible to genotype high numbers of samples rapidly and accurately. We analysed digestive trypsins of two Daphnia magna populations as an application example for HRMA. One population originated from a pond containing toxic cyanobacteria that possibly produce protease inhibitors and the other from a pond without such cyanobacteria. The hypothesis was that D. magna clones from ponds with cyanobacteria have undergone selection by these inhibitors, which has led to different trypsin alleles. We first sequenced pooled genomic PCR products of trypsins from both populations to identify variable DNA sequences of active trypsins. Second, we screened variable DNA sequences of each D. magna clone from both populations for single nucleotide polymorphisms via HRMA. The HRMA results revealed that both populations exhibited phenotypic differences in the analysed trypsins. Our results indicate that HRMA is a powerful genotyping tool for studying the variation of target genes in response to selection within and between natural Daphnia populations.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Kuster, C. J. UNSPECIFIED UNSPECIFIED UNSPECIFIED Von Elert, E. UNSPECIFIED UNSPECIFIED UNSPECIFIED
URN:	urn:nbn:de:hbz:38-480134
DOI:	10.1111/j.1755-0998.2012.03177.x
Journal or Publication Title:	Mol. Ecol. Resour.
Volume:	12
Number:	6
Page Range:	S. 1048 - 1058
Date:	2012
Publisher:	WILEY-BLACKWELL
Place of Publication:	HOBOKEN
ISSN:	1755-098X
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language SINGLE NUCLEOTIDE POLYMORPHISM; TRANSCRIPTIONAL ACTIVITY; MICROCYSTIS-AERUGINOSA; TOXIC CYANOBACTERIA; PROTEASE INHIBITORS; DIVERSITY; BINDING; PULEX; DIFFERENTIATION; SUBSTITUTION Multiple languages Biochemistry & Molecular Biology; Ecology; Evolutionary Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/48013