Abstract
In order to anchor Rosa linkage groups to physical chromosomes, a combination of the Tyramide-FISH technology and the modern molecular marker system based on High Resolution Melting (HRM) is an efficient approach. Although, Tyramide-FISH is a very promising technique for the visualization of short DNA probes, it is very challenging for plant species with small chromosomes such as Rosa. In this study, we successfully applied the Tyramide-FISH technique for Rosa and compared different detection systems. An indirect detection system exploiting biotinylated tyramides was shown to be the most suitable technique for reliable signal detection. Three gene fragments with a size of 1100 pb–1700 bp (Phenylalanine Ammonia Lyase, Pyrroline-5-Carboxylate Synthase and Orcinol O-Methyl Transferase) have been physically mapped on chromosomes 7, 4 and 1, respectively, of Rosa wichurana. The signal frequency was between 25% and 40%. HRM markers of these 3 gene fragments were used to include the gene fragments on the existing genetic linkage map of Rosa wichurana. As a result, three linkage groups could be anchored to their physical chromosomes. The information was used to check for synteny between the Rosa chromosomes and Fragaria.
Highlights
Genome structure and function may be studied when comparing the genetic positions of genes with their physical locations on chromosomes
A more efficient approach exists by direct visualization of genetically mapped markers on chromosomes using fluorescent in situ hybridization (FISH) to locate large genomic clones (BAC, YAC, cosmids etc.) containing the markers
In the indirect detection with two rounds of amplification system, signals for PAL became visible under the following conditions: a first round using streptavidin-horseradish peroxidase (SA-Horse Radish Peroxidase (HRP)) (1:100), Tyr-Bio (1:25), 5 min tyramide incubation time and a second round using SA-HRP (1:300), Tyr-Cy3 (1:500), 6 min tyramide incubation time
Summary
Genome structure and function may be studied when comparing the genetic positions of genes with their physical locations on chromosomes. In former times, to assign linkage groups to physical chromosomes it was needed to create monosomic addition lines, nullisomic lines, chromosome substitution lines or translocation lines [1,2,3]. FISH with large genomic DNA fragments often results in many non-specific hybridization due to the presence of huge amounts of repetitive DNA in plant genomes [4,5]. To overcome this problem, FISH using direct labeled individual genes can be applied [6,7,8]. This approach still is very challenging for most ornamental species and in particular for woody species, such as Rosa
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