The taxonomic classification of the sampled plants in the field was based on morphology characteristics, in addition, two other clover species (T. repens and Trifolium hybridum) also grow in those locations and can look similar to T. pratense. As the sampling had to be performed in a short period of time, I decided to verify the taxonomic identity of the samples using a DNA barcoding approach. For this reason I developed a DNA barcoding method based on the internal transcribed spacer (ITS) (ITS4 and ITS5) region for T. pratense plants, to verify the identity of the collected plants. The ITS region is very suitable as a barcode for plants and to identify plants at a species level, as it is easy to amplify and shows a high degree of variation in addition it was used in previous studies to determine phylogenetic relationships within the Trifolium genus (Ellison et al. 2006; Watson et al. 2000) The barcoding method is based on the amplification followed by the digestion of the ITS fragment of the T. pratense plants. Thereby the ITS fragment is cut into pieces of different sizes, which is species specific due to different restriction sites because it is a variable region. In preparation the sequence of the ITS fragments for T. pratense, T. repens and T. hybridum were digitally digested with the program Bioedit (Hall 1999) with several restriction enzymes to identify which band pattern (size and number of the bands) we could expect for each clover species (table 4). Based on those results (table 4) an enzyme was selected. We choose MseI, this cannot distinguish between T. repens and T. hybridum, but it is possible to see a clear separation between T. pratense and the both other clover species. DNA was extracted from T. pratense and T. repens plants, followed by a PCR. The product gained from the PCR reactions were digested using MseI. Afterwards, the digested PCR products were applied on an agarose gel and the samples were identified based on the pattern on the gel.
3.3.1. DNA barcoding approach: RFLP of plastid ITS region
For the laboratory work the samples collected in the field were used (in total 32 samples) and two samples clearly identified as T. pratense and T. repens. DNA was extracted using the quick and dirty DNA extraction as described in (Weigel and Glazebrook 2002). For the amplification of the ITS region Primer ITS4 and ITS5 (White et al. 1990) were selected, producing an approximately 689 bp long fragment for T. pratense and T. repens. A PCR was performed with two individuals of good DNA quality and known taxonomic identity to check the specificity of the primers and optimize the PCR protocol. Therefore, two individuals of good DNA quality were used, as a positive control. Additionally, one negative control was included in each run to detect contamination. The PCR was optimized and then conducted with the collected clover species, based on the following master mix (table 2).
26 Table 2 Master mix for PCR reactions of ITS region of T. pratense and T. repens DNA
Reagent Volume per reaction
ddH2O 39.2μl
Forward primer (10μM) 1.25μl
Reverse primer (10μM) 1.25μl
Buffer (10x Dream Taq)* 5μl
dNTPs (10mM)* 1µl
Taq (5U/µl)** 0.3µl
DNA 2µl
Total volume 50µl
*Thermo Scientific,Frankfurt
**Dream Tag DNA Polymerase,Thermo scientific,Frankfurt
The standard PCR was a set of standard PCR cycles followed (denaturation, annealing, and elongation, table 3).
Table 3 Cycler* settings for PCR reactions of ITS region of T. pratense and T. repens DNA
Cycler settings Temperature Duration Initial denaturation 94˚ 4 min
Standard cycle 35 cycles
Denaturation 94˚ 30 sec
Annealing 55° 30 sec
Elongation 72˚ 1 min
Final Elongation 72˚ 7 min
* Biometra
The success of the PCR amplification, was evaluated by agarose gel electrophoresis. This made it possible to determine the positive and negative controls to exclude false positive or false negative results due to contamination issues. For this purpose a 1.5% agarose (Biozym LE Agarose, Oldendorf, Germany) solution based on TAE (Tris-Acidic Acid-EDTA) buffer (1X) was prepared and 2 µl of the DNA stain DNAstainG™ (Serva, Heidelberg, Germany) per 100 ml gel were added according to the instruction manual (relation 1:20). For each gel, approximately 50 ml of 1.5% agarose gel solution was used. After the agarose solidified, the chamber was filled with 1x TAE buffer until the gel was covered. During the next step the gel slots were loaded with a mix of 3 μl PCR product and 1 μl (1x) loading buffer (Thermo Scientific 6X DNA Loading Dye, Thermo Scientific, Frankfurt) with an exception of one slot, loaded with a DNA ladder (MassRuler™DNA Ladder Thermo Scientific, Frankfurt). Agarose gels were run at 85 V for 35 min. Pictures were taken under UV light (Biorad, Hercules, California), to display the DNA, intercalating with DNAstainG™ (Serva, Heidelberg, Germany).
27 The PCR template of T. pratense and T. repens was analyzed in a RFLP analysis with MseI. During this reaction the PCR template is cut in pieces of different number and size to enable a species specific taxonomic classification. The digest reaction (table 5) was optimized and adjusted with the two individuals of T. pratense and T. repens. After the optimal conditions were found all sampled field individuals were analyzed.
Table 4 Expectation of the fragment size and number of fragments after the digestion with MES I for three clover species (T. pratense, T. repens, T. hybridum). The smallest fragment (50 bp) of T. pratense is set in brackets due to its small size, as it will not be possible to see it during the gel documentation. Anyways it is not necessary for distinguish against the two other clover species.
Species fragment size Number of fragments
T. pratense (50),120,200,370 3(4)
T. repens 150,450 2
T. hybridum 200,470 2
Table 5 Master mix for Digestion reactions* with MseI of PCR template of T.pratense and T. repens
Regent Volume per reaction
MseI (1U)** 1μl Cutsmart Buffer (10x)** 2μl ddH2O 14.8μl Template 2.2μl Total volume 20µl *Incubated at 37°C for 1h **NEB, Frankfurt
The success of the digest reaction, meaning the number and the sizes of the digestion, so the expected cutting patterns, was evaluated by agarose gel electrophoresis (for details see section above).