Title: Detection of Sequence Polymorphism in Rubus Occidentalis L. Monomorphic Microsatellite Markers by High Resolution Melting Authors
Submitted to: International Horticultural Congress
Publication Type: Abstract Only
Publication Acceptance Date: March 31, 2010
Publication Date: N/A
Interpretive Summary: We have been trying to identify different types of black raspberries using DNA fingerprinting techniques. One technique that we used very successfully to identify and distinguish many named blueberries (cultivars) was called “simple sequence repeats,” abbreviated as SSRs. The SSRs involved finding bits of DNA that had different lengths of tandemly repeated sections of two to three letter codes. These made different banding patterns and allowed us to separate ‘Duke’ blueberry from ‘Bluecrop’ blueberry, for example. Sometimes the differences in these SSRs are in the type of code and not in the length of the DNA. For those SSRs, we tried a new technique that involves melting the fluorescently labeled DNA at high temperature and observing the profile or ‘melting curve’ of the DNA as the fluorescence of the DNA decreases with increasing temperatures. This new technique is called “high resolution melting” or HRM. The HRM technique lets us look at a section of DNA from one cultivar, compare it to similar sections of DNA in other cultivars, and differentiate between them based on only one single difference in the sequence. Using the new technique we could separate black raspberry cultivars that we did not separate using the SSR technique. We bred or crossed two types of black raspberry and could look at how this single code difference was inherited in offspring. This new technique will be very useful to help us map genes onto the DNA codes for black raspberries and other crops.
Technical Abstract: Microsatellite, or simple sequence repeat (SSR) markers, are valuable as co-dominant genetic markers with a variety of applications such as DNA fingerprinting, linkage mapping, and population structure analysis. Development of microsatellite primers through the identification of appropriate repeated sequences and flanking regions often leads to a high proportion of these markers that are monomorphic and of little value. The development of high resolution melting (HRM) technologies provides a new method of identifying sequence variations, such as single nucleotide polymorphisms (SNPs), in monomorphic SSR products. In this study, we used HRM to identify SNPs in monomorphic PCR products generated with SSR primer pairs in the parents of a black raspberry mapping population. HRM of PCR amplicons, using these same primer pairs, was then used to genotype seedlings in this mapping population. The observed markers segregated in a Mendelian fashion based on the genotypes of the parent plants. HRM can be a valuable tool for detecting and adding SNP markers to genetic linkage maps using existing primers that are otherwise unusable.