Background: Molecular phylogeny allows to assign a specimen to a specific position in the tree based on the sequence of a marker gene. Especially for so called barcoding markers, the public databases provide meanwhile a large number of sequences. However, the authentication is time consuming, because the marker has to be amplified by PCR and sequenced, before the potential identity of the sample can be assessed through alignment with known sequences. In many cases, the practical application is to test a given sample for the authentity of the species declared and to safeguard against so called adulteration by other species (for instance cheap surrogates or, even worse, hazardous material that was mixed up for different reasons). Here, assays that allow to see the identity at once, are warranted. These assays are usually based upon so called genetic fingerprints. The strategy is to design a PCR-based approach that will yield different banding patterns that can then be easily assigned to the species of interest and that will clearly differ in case of adulteration. In the lab, we use this approach for novel plant species that are now fashionable in consequence of new food trends (for instance in the health sector). Globalisation has brought here considerable challenges with new products entering the European market at high speed. Most of these products come from different medical traditions that differ, also with respect to nomenclature, such that numerous misunderstandings, misspellings, and also deliberate surrogations are frequent. A specific subgroup of these fingerprints, so called microsatellites, allow to distinguish even individual genotypes of a population, and are useful, when the phylogenetic relationships within a species have to be assessed. Specifically, the domestication of crop plants from a wild ancestor, can be assessed by such microsatellites (see next lecture).
Task: one of these fingerprinting techniques uses small differences in sequence, for instance single nucleotide polymorphisms (SNPs) to generate a different banding pattern making use of restriction enzymes that either cut or do not cut, depending on presence or absence of the SNP. This approach is called Restriction Fragment Length Polymorphism (RFLP) and belongs to the DNA fingerprinting techniques. During a study on the origin of Weedy Red Rice in Italy, we observed that there exist two groups for this weed making use of the sequence of Rc (a transcription factor that regulates anthocyanin synthesis in the pericarp of the caryopsis). While cultivated rice harboured a 14-bp deletion, the weedy rice was forming two populations. Population 1 did not how this deletion (meaning that this allele was coming from a wild ancestor), whereas population 2 did show the deletion, but in addition carried nearby a second deletion of 1 bp. Since 1+14 gives 15, this second site mutation restored the reading frame such that a slightly smaller, but functional version of the protein resulted. This second group obviously evolved from cultivated rice by a second site mutation. In a field study, we had collected several hundreds of these samples and wanted to clarify, whether they are of type 1 or of type 2. To sequence the Rc gene for all of them would be expensive and time consuming. So, we designed a RFLP strategy to assign each individual to one of the two groups.
- based on the alignment of the three types (right-click here and download the alignment) design a digest strategy. The entire fragment (the borders are not shown on the alignment) is 500 bp
- find out, which restriction enzymes can be used to distinguish the two alleles (with and without the 1 bp deletion) and what fragments would result
Advice: in the web, there exist several sites, where restriction sites of a sequence can be determined (for instance here).
The slides for this task, along with a recorded lecture on this task are available on Ilias. Note: The lecture slides can be found on Ilias (log in with your KIT credentials, if you are outside the KIT, make sure that you are linked through vpn). Path: Fakultät für Chemie und Biowissenschaften - current semester - BIO_MA_FOR_1201_Plant_Evolution)
The paper is available through the KIT web:
- one of our publications where we use RFLP to discriminate two equally named species ("Lemon Myrtle") - pdf