Development and application of high-throughput amplified fragment length polymorphism technique in Calluna vulgaris (Ericaceae)

Abstract

Calluna vulgaris is an important ornamental crop of the horticultural industry in Europe. In order to improve breeding of this species, especially of the most important trait of 'bud-flowering', the implementation of molecular techniques that allow rapid, reproducible and efficient screening of whole segregating populations e.g. for molecular marker and mapping approaches is a requirement. We therefore aimed to introduce the powerful tool of amplified fragment length polymorphisms (AFLP ® ), a widely and successfully applied method, into our methodological assortment. As an essential prerequisite, the isolated DNA should be of adequate quality which is a common obstacle when dealing with woody species and their interfering secondary components/metabolites. The results of screening different and modified DNA isolation protocols are described. As the outcome of our evaluations of reaction conditions during the AFLP ® procedure, we circumstantiate a functional protocol ranging from DNA extraction to visualization of AFLP ® banding patterns for the woody crop C. vulgaris. This method is suitable for high throughput genetic applications and may even be transferable to other species. In addition, costs are reduced by reasonable reagents and multiplexing assays. *Corresponding authorRecently, we reported a DNA isolation method for the ornamental crop C. vulgaris suitable for randomly amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) analyses (Borchert et al. 2008). However, this method incorporates potentially harmful phenol/chloroform extraction steps and requires approximately 14 hrs of working time for 24 samples (from tissue grinding to quantification and qualification). Moreover, RAPD and ISSR techniques are not adequate e.g. regarding reproducibility especially for mapping of traits. Therefore, we aimed at improving our method by simplifying the DNA isolation protocol for C. vulgaris and establishing amplified fragment length polymorphisms (AFLP ® ) methodology. Both methods should comply with high-throughput (htp) requirements in terms of time-and cost-efficiency. AFLPs® require high-quality DNA. The isolation of such DNA of herbaceous and woody plants is commonly hindered due to the accumulation of polyphenolic and polysaccharide compounds that interfere during subsequent processing of the DNA. Several manual protocols based on different assays to eliminate these contaminants (e.g. CTAB, silica; (Rogstad, 2003)) are available for such cases. However, for each species the adaptation of the isolation method is usually required.

AFLP-based genetic mapping of the “bud-flowering” trait in heather (Calluna vulgaris)

“…vulgaris was found to be optimal with double-digestion by HindIII and MseI, a preamplification using non-selective and non-labelled primers and selective amplification with three selective bases at the 3′-end of the HindIII and MseI primers [10]. HindIII and MseI primer (+3/+3) or (+2/+3) combinations resulting in most polymorphic bands were chosen from the pre-test for the mapping approach and their reproducibility was proven.…”

“…The AFLP procedure was conducted and its reproducibility tested according to [10]. MseI, HhaI, and HindIII were used to digest diluted DNA.…”

“…vulgaris is very limited and the AFLP (Amplified Fragment-Length Polymorphism) procedure has already been adapted for C . vulgaris [10]. Hence, AFLPs have been chosen for the rapid generation of markers covering the genome.…”

Molecular Reconstruction of an Old Pedigree of Diploid and Triploid Hydrangea macrophylla Genotypes

“…The PCR conditions were 3 min at 94 °C, 35 cycles of 30 s at 94 °C, 30 s at 60 °C and 30 s at 72 °C, and finally 5 min at 72 °C. PCR fragment lengths were determined by polyacrylamide gel electrophoresis according to Borchert and Gawenda ( 2010 ). For this, we mixed 5 μl IRD 700 PCR product, 5 μl IRD 800 PCR product and 90 μl pararosaniline loading dye and separated PCR fragments together with a 50–700 bp sizing standard (LI-COR Biosciences) on a 6.5% KB Plus gel matrix (LI-COR Biosciences) using a LI-COR 4300 DNA analyzer (LI-COR Biosciences).…”