Molecular identification of fine roots of trees from the Alps: reliable and fast DNA extraction and PCR–RFLP analyses of plastid DNA

Abstract

Fine roots of trees are intensively used as indicators to assess soil alterations, e.g. those owing to atmospheric inputs of acidifying substances, but their identification to species with morphological criteria is difficult. In this study, we established molecular techniques in order to identify fine roots of the 30 most common tree species of the Alps. We developed a protocol for efficient isolation of DNA from fine roots with extraction of DNA in the presence of polyvinylpyrrolidone (PVP) and polyvinylpolypyrrolidone (PVPP). The trnL (UAA) intron of plastid DNA was used as a marker for fine root identification. We amplified and sequenced this intron with plant universal primers. The size of the sequences ranged from 444 to 672 bp. A synoptic key for species identification was designed on the basis of restriction fragment patterns predicted from sequence data. Using the restriction enzyme TaqI as key enzyme, and where necessary HinfI, RsaI and CfoI, 16 taxa, including Picea abies, Larix decidua, Abies alba, and Fagus sylvatica, the dominant tree species of the Alpine region could be identified by agarose gel electrophoresis of restriction fragments. Fourteen taxa could be identified to the genus level, among them Quercus, Salix and Populus species. In a field study, conducted in a 20 x 30 m plot of a mixed forest with five tree species, fine roots of 43 out of 46 samples were identified and their distributions were mapped. These results demonstrate the utility of our DNA extraction method and of the trnL intron for the identification of fine tree roots.

Effective DNA extraction method for fragment analysis using capillary sequencer of the kelp, Saccharina

“…It is known that polyvinylpyrrolidone (PVP) adsorbs the polyphenol and β-mercaptoethanol shows an antioxidative effect (e.g. Porebski et al 1997;Dempster et al 1999;Khanuja et al 1999;Brunner et al 2001;Biss et al 2003;Bharmauria et al 2010;Kejani et al 2010). In the present study, the extraction solution with the modified CTAB method contained PVP and β-mercaptoethanol, and it is suggested that this DNA extraction technique is effective both to remove the polyphenol and to curb the oxidization of polyphenol.…”

Belowground DNA-based techniques: untangling the network of plant root interactions

“…Jones et al (2011), therefore, used trnH-psbA to discriminate between 33 species from 117 root fragments. However, in recent root studies, an alternative, and often less species specific, barcode (trnL) is more frequently used (Brunner et al 2001;Ridgway et al 2003;Frank et al 2010;Dumbrell et al 2010;Taggart et al 2011). This variety in the usage of different barcode primers reflects that primer choices will always need to be adjusted to the molecular differentiation found across species within a particular community or experimental system.…”

Identifying roots of northern hardwood species: patterns with diameter and depth

“…The species of root branches <2 mm in diameter were identified with molecular genetic methods using the procedure described by Brunner et al (2001). We analyzed 2-5 root branches from the surface layer of each of the six pit samples at Bartlett.…”

“…Root samples were ground in an amalgamator (Darby Dental, Akron, Ohio). DNA was extracted from approximately 30 mg of each sample using a standard alkaline lysis or chloroform extraction procedure modified with addition of polyvinylpolypyrrolidone, polyvinylpyrrolidone, betamercaptoethanol, and spermidine, to improve extraction efficiency and inhibitor removal (Brunner et al 2001). The plastid trnL intron was amplified using primers c and d (Taberlet et al 1991) and PCR products were digested with taqI.…”