Molecular markers - SNPs: Single Nucleotide Polymorphisms
SNPs (pronounced “snips”) are differences in DNA sequence of just one (or sometimes a small number of) nucleotides. Where these differences occur within a genic sequence, they are more often than not phenotypically neutral, but sometimes they can be associated with a change in the amino acid sequence of the gene product. They are very common, and are distributed throughout the genome. SNP genotyping can be relatively simple, but SNP discovery generally requires extensive DNA sequencing. However, because much of the procedure is automated, the price of SNPs is much less than earlier types of markers including SSRs, so SNPs now dominate the molecular breeding field.
Genotyping is the process of determining the genetic constitution-the genotype-of an individual plant by examining their DNA sequence using biological assays and comparing it to another individual’s sequence or a reference sequence.
Plant phenotyping is the comprehensive assessment of plant complex traits such as growth, development, tolerance, resistance, architecture, physiology, ecology, yield, and the basic measurement of individual quantitative parameters that form the basis for the more complex traits. Examples for such direct measurement parameters are image-based projected leaf area, chlorophyll fluorescence, stem diameter, plant height/width, compactness, stress pigment concentration, tip burn, internode length, colour, leaf angle, leaf rolling, leaf elongation, seed number, seed size, tiller number, flowering time, germination time etc[.: www.lemnatec.com/plant-phenotyping]
Figure 9. The figure below is an example of a SNP between 2 small DNA sequences. The sequences are the same order of nucleotides except for the one in red. This SNP would be designated as G:T, since one organism has a G (guanine) at this location of the genome while the other organism has a T (thymine).
For a review of “next generation” sequencing technologies, see: Shendure and Hanlee 2008.
One SNP at a time
High-throughput systems are based on high-throughput! Figure 9 shows typical plates that are used for SNP genotyping, which can hold 96 or 384 samples at once. Therefore you generally can’t run just one SNP marker at a time. However a SNP can be converted into a single use marker for Marker Assisted Breeding by a number of techniques, such as CAPs (Cut/Cleaved Amplified Polymorphism).
Figure 9a: Plates used for SNP genotyping. Image from http://www.wellplate.com/the-history-of-the-microplate/.
If a SNP falls on an enzyme restriction site (Restriction sites, or restriction recognition sites, are locations on a DNA molecule containing specific (4-8 base pairs in length sequences of nucleotides, which are recognized by restriction enzymes) , primers can be designed so that a simple PCR assay discriminates the alleles following an enzyme digest. This process converts a SNP that is part of a larger assay to one that can be genotyped individually. Some DNA sequence information is needed to create such an assay, however. But this can be very useful for a marker that has special importance, for example one that is tightly linked to a disease resistance gene. Figure 10, from Varshney et.al. 2007, is a gel showing 12 chickpea lines genotyped with a CAPS assay developed for a SNP marker. In this case, the scientists used DNA sequence information previously identified with ESTs (expressed sequence tags) to pinpoint a region that could be differentiated with a restriction enzyme; then, primers were designed to amplify that region. This new marker shows polymorphism in line 5, differentiating that line from the other chickpea lines.
See the Varshney et al. ICRISAT publication for more information (Varshney RK, Nayak S, Jayashree B, Eshwar K, Upadhyaya HD, Hoisington DA: Development of cost-effective SNP assays for chickpea genome analysis and breeding. SAT eJournal 2007, 3:1-3.)
Figure 10: A CAPS assay genotype of chickpea lines. Image from Varshney et.al. 2007.
ARMS Tetra-primer system
The amplification refractory mutation system (ARMS) is another method of creating an assay for one particular SNP. This uses an amplification strategy in which polymerase chain reaction (PCR) primers are designed in such a way that they are able to discriminate among templates that differ by a single nucleotide, thus converting a SNP into a simple PCR assay, albeit generally one with 4 primers. The trial-and-error procedure to develop an assay in this way can be time-consuming, so is used only for markers of special importance.