ARS | Publication request: High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: August 1, 2011
Publication Date: January 1, 2012
Citation: Dossett, M., Bassil, N.V., Finn, C.E. 2012. High resolution melting detects sequence polymorphism in rubus occidentalis L. monomorphic microsatellite markers. Acta Horticulturae. 926:91-95.

Interpretive Summary: Microsatellites or simple sequence repeats (SSRs) are DNA markers with a variety of applications for genetic studies. Methods used to develop microsatellite primers often lead to a high proportion of markers with no apparent size differences and little value. The development of high resolution melting (HRM) technologies provides a new method of identifying DNA sequence variations, such as single nucleotide polymorphisms (SNPs), in these amplified DNA fragments. In this study, we used HRM to identify SNPs using SSR primer pairs in two black raspberry selections. Using these same primer pairs, HRM was then used to determine the inheritance of these SNPs in 22 progeny from the cross of these two black raspberry selections. HRM can be a valuable tool for detecting and adding SNP markers to genetic maps with existing primers that are otherwise unusable.

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. However, primer pairs designed from the regions that flank SSRs often generate fragments with no size polymorphism. High resolution melting (HRM) technology provides a new method of identifying sequence variations in these amplified fragments. In this study, we used HRM to identify polymorphism in SSR PCR products that were monomorphic for size in a black raspberry (Rubus occidentalis L. subgenus Idaeobatus) population. When HRM revealed polymorphism between the parents of a mapping population, the seedlings were genotyped and scored. The HRM markers segregated in a Mendelian fashion. Comparison of DNA sequences of the parents revealed the presence of single nucleotide polymorphisms (SNP) in the amplicons. HRM can be a valuable tool for detecting polymorphism in PCR amplicons using existing primer pairs that would otherwise be of little use.