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

Kirov et al. BMC Genetics (2015) 16:74 DOI 10.1186/s12863-015-0233-9 RESEARCH ARTICLE Open Access High resolution physical mapping of single gene fragments on pachytene chromosome 4 and 7 of Rosa Ilya V. Kirov1,2,3, Katrijn Van Laere3* and Ludmila I. Khrustaleva1,2 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. Keywords: Fluorescence In Situ Hybridization, Pachytene, Tyramide-FISH, Rosa, Physical map Background Rosa is a genus of the Rosaceae family consisting of approximately 90 genera and approximately 3000 species. Many of these are economically important such as Malus, Prunus, Pyrus, Fragaria, Rubus, Sorbus, Cotoneaster and Crataegus [1–5]. Approximately 150 species and more than 20.000 cultivars of Rosa are described [6]. Most species have a complex origin [7]. Interestingly, only 7 to 15 species have contributed to the original germplasm of the modern rose cultivars [8]. Rosa species have small genomes and a high level of heterozygosity. Basic chromosome number is x = 7 [1], but * Correspondence: 3 Plant Sciences Unit, Applied Genetics and Breeding, Institute for Agricultural and Fisheries Research (ILVO), Caritasstraat 21, 9090 Melle, Belgium Full list of author information is available at the end of the article ploidy levels range from diploid (2n = 2x = 14) to decaploid (2n = 8x = 56). Genomes of Pyrus [9], Prunus [10], Fragaria [4] and Malus [11] recently have been sequenced, providing valuable information for comparative genomics, gene cloning, marker development, QTL mapping and marker-assisted selection. Comparative analysis of these sequenced genomes has shed more light on the mode of evolution of some Rosaceae genera and species. In contrast, the organization of the Rosa genome has only been poorly investigated and the knowledge about the macro-synteny and collinearity of the Rose genome with other sequenced genomes Rosaceae family is poor. Genetic maps have been widely used for comparative genomic and genome organization studies [12, 13]. The distance between markers in genetic maps expressed in © 2015 Kirov et al. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http:// creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Kirov et al. BMC Genetics (2015) 16:74 recombination frequencies, or centimorgans (cM) is known to be unequally distributed along the chromosomes [14–17]. Gene order in genome regions with extremely low recombination frequency (e.g. centromeres, heterochromatin) cannot be revealed because of the low resolution of genetic mapping in these regions [18]. In addition, lack of collinearity between parental genomes used for development of the mapping population can cause inaccuracy in genetic maps [19]. In contrast to genetic maps, physical maps show real positions of DNA sequences on the chromosomes. Physical mapping using fluorescence in situ hybridization (FISH) does not depend on recombination frequency, therefore it can be used for gene mapping in “cold spot recombination” regions [18]. But FISH mapping has a lower efficiency than genetic mapping. The integration of physical and genetic maps provides a unique tool combining advantages of both types of maps. FISH-based physical maps have been developed and successfully integrated with genetic maps for many plant species (see review [20]). Direct visualization of DNA sequences on chromosomes by FISH is also a valuable for genome sequencing. FISH mapping improves the quality of genome assembly as demonstrated in tomato [21], cucumber [22] and Amborella [23]. Until now most of the cytogenetic studies in Rosa have been dedicated to karyotyping, chromosome number evaluation and rRNA (5S and 45S) gene mapping [24–28]. Further progress in FISH using individual genes or DNA clones (e.g. ESTs, BACs) is required for efficient cytogenetic map construction. Physical mapping of individual genes as small as 1.1 kb has proven to be possible on mitotic metaphase chromosomes of Rosa wichurana using TyramideFISH [29]. However, the resolution of Tyramide-FISH on the small mitotic Rosa chromosomes is very low which significantly hampers the construction of a physical map and the determination of the order of DNA sequences. The use of pachytene chromosomes would be an improvement for physical mapping [30]. Pachytene chromosomes are 7–40 times longer than mitotic metaphase chromosomes and therefore provide a higher resolution [21, 30]. Moreover, heterochromatic and euchromatic regions are distinguishable at the pachytene stage [31, 32]. Pachytene bivalents consist of 8 DNA strands instead of 4 in mitotic chromosomes, which also increases the sensitivity of FISH. Also important is that meiotic cells (pollen mother cells, or PMC) synchronously divide providing many cells in the same stage. High resolution FISH mapping on pachytene chromosomes has been used successfully in tomato [14, 21, 33–35] a (...truncated)