High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa

Abstract

Background: Rosaceae is a family containing many economically important fruit and ornamental species. Although fluorescence in situ hybridization (FISH)-based physical mapping of plant genomes is a valuable tool for map-based cloning, comparative genomics and evolutionary studies, no studies using high resolution physical mapping have been performed in this family. Previously we proved that physical mapping of single-copy genes as small as 1.1 kb is possible on mitotic metaphase chromosomes of Rosa wichurana using Tyramide-FISH. In this study we aimed to further improve the physical map of Rosa wichurana by applying high resolution FISH to pachytene chromosomes.

Results: Using high resolution Tyramide-FISH and multicolor Tyramide-FISH, 7 genes (1.7-3 kb) were successfully mapped on pachytene chromosomes 4 and 7 of Rosa wichurana. Additionally, by using multicolor Tyramide-FISH three closely located genes were simultaneously visualized on chromosome 7. A detailed map of heterochromatine/euchromatine patterns of chromosome 4 and 7 was developed with indication of the physical position of these 7 genes. Comparison of the gene order between Rosa wichurana and Fragaria vesca revealed a poor collinearity for chromosome 7, but a perfect collinearity for chromosome 4.

Conclusions: High resolution physical mapping of short probes on pachytene chromosomes of Rosa wichurana was successfully performed for the first time. Application of Tyramide-FISH on pachytene chromosomes allowed the mapping resolution to be increased up to 20 times compared to mitotic metaphase chromosomes. High resolution Tyramide-FISH and multicolor Tyramide-FISH might become useful tools for further physical mapping of single-copy genes and for the integration of physical and genetic maps of Rosa wichurana and other members of the Rosaceae.

Fig. 1

Pachytene (a), Metaphase I (b) and Tetrad (c) stages found in a R. wichurana flower bud. Bars = 10 μm

Fig. 2

Inverted DAPI pictures and ideogram of chromosome 4 (a) and 7 (b). Red signals showed the location of AAA-2 (a) and MGM (b) genes after Tyramide-FISH to verify the correct chromosome number. Stars indicate the centromere position on the chromosomes

Fig. 3

In situ physical mapping of genes on pachytene chromosomes of Rosa wichurana. FISH with Dig-labeled 45S rDNA (pTA71 plasmid) (a); Tyramide-FISH with MLO3 and P5CS genes, both labeled with biotin (b) and with AAA-2 gene, labeled with biotin (c); Tyramide-FISH with MGM gene labeled with biotin (d) and MDAR gene labeled with biotin (e); Sequential multicolor Tyramide-FISH with digoxigenin labeled MLO2 gene and biotin labeled PAL and MGM genes (f). Centromere of the chromosome that contains the physically mapped gene(s) is indicated by an arrowhead. Bar = 10 μm

Fig. 4

Physical location of 7 target genes on R. wichurana chromosome 7 (RwChr7, left) and 4 (RwChr4, right) versus Fragaria vesca pseudochromosomes 6 (FvChr6) and 7 (FvChr6), correspondently. Signals are shown on digitally straightened pachytene chromosomes and on an ideogram of R. wichurana and compared with pseudochromosome 6 (FvChr6) and 7 (FvChr7) of the F. vesca genome sequence