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

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

Towards a FISH-based karyotype of Rosa L. (Rosaceae)

The genus Rosa Linnaeus, 1753 has important economic value in ornamental sector and many breeding activities are going on supported by molecular studies. However, the cytogenetic studies of rose specks are scarce and mainly focused on chromosome counting and chromosome morphology-based karyotyping. Due to the small size of the chromosomes and a high frequency of polyploidy in the genus, karyotyping is very challenging for rose species and requires FISH-based cytogenetic markers to be applied. Therefore, in this work the aim is to establish a FISH-based karyotype for Rosa wichurana (Crepin, 1888), a rose species with several benefits for advanced molecular cytogenetic studies of genus Rosa (Kirov et al. 2015a). It is shown that FISH signals from 5S, 45S and an Arabidopsis-type telomeric repeat are distributed on five (1, 2, 4, 5 and 7) of seven chromosome pairs. In addition, it is demonstrated that the interstitial telomeric repeat sequences (ITR) are located in the centromeric regions of four chromosome pairs. Using low hybridization stringency for ITR visualization, we showed that the number of ITR signals increases four times (1-4 signals). This study is the first to propose a FISH-based R. wichurana katyotype for the reliable identification of chromosomes. The possible origin of R wichurana ITR loci is discussed

Hybridization of the RT-CR probes on chromosomes of <i>D</i>. <i>villosum</i>, <i>P</i>. <i>spicata</i>, <i>Th</i>. <i>bessarabicum</i>, and <i>Th</i>. <i>intermedium</i>.

<p><b>a-c</b> FISH analysis using RT-CR probes on the root-tip cells at mitotic metaphase in a) <i>D</i>. <i>villosum</i>, b) <i>P</i>. <i>spicata</i>, c) <i>Th</i>. <i>bessarabicum</i>; the metaphase plates are probed with <i>Davi</i> (a, green), <i>Pssp</i> (b, pink) and <i>Thbe</i> (c, pink). Bar = 5 μm. <b>d-f</b> Sequential multicolor FISH and multicolor GISH on the root-tip cells at mitotic metaphase in <i>Th</i>. <i>intermedium</i>: d) the mcFISH results using the <i>Thbe</i> probe (red); e) the same cell in (d) with <i>Pssp</i> probe (green); f) GISH analysis for the same cell in (d) and (e) with the labeled genomic DNA of <i>P</i>. <i>spicata</i> (green) and <i>D</i>. <i>villosum</i> (pink) as probes and <i>T</i>. <i>aestivum</i> genomic DNA (ABD genome) as a block. Bar = 10 μm. <b>g-i</b> Karyotype of chromosomes of <i>Th</i>. <i>intermedium</i> from (d-f) with the results of sequential multicolor FISH and multicolor GISH. Chromosomes organized into genomes J^r, J^vs and St according to St- and V-genome DNA labeling and FISH signal intensity in centromeric region. The J^vs chromosomes grouped into J^v chromosomes (in frame) and J^s chromosomes. The translocated chromosome marked with asterisk. g) Chromosomes with <i>Thbe</i> probe (red); h) The same chromosomes with <i>Pssp</i> probe (green); i) The same chromosomes labeled with genomic DNA of <i>P</i>. <i>spicata</i> (St genome, green) and <i>D</i>. <i>villosum</i> (V genome, pink) as probes and <i>T</i>. <i>aestivum</i> genomic DNA (ABD genome) as a block. Chromosomes counterstained with DAPI (blue).</p

The dendrogram of the deduced RT-CR amino acid sequences and their homologs from the NCBI Nucleotide collection search database.

<p>The dendrogram was constructed using the Maximum Likelihood method based on the JTT matrix-based model. The bootstrap consensus tree inferred from 1000 replicates. The colored bullets indicate the RT-CRs obtained in the present study.</p

Variation in Copy Number of Ty3/Gypsy Centromeric Retrotransposons in the Genomes of <i>Thinopyrum intermedium</i> and Its Diploid Progenitors

<div><p>Speciation and allopolyploidization in cereals may be accompanied by dramatic changes in abundance of centromeric repeated transposable elements. Here we demonstrate that the reverse transcriptase part of Ty3/gypsy centromeric retrotransposon (RT-CR) is highly conservative in the segmental hexaploid <i>Thinopyrum intermedium</i> (J^rJ^vsSt) and its possible diploid progenitors <i>Th</i>. <i>bessarabicum</i> (J^b), <i>Pseudoroegneria spicata</i> (St) and <i>Dasypyrum villosum</i> (V) but the abundance of the repeats varied to a large extent. Fluorescence <i>in situ</i> hybridization (FISH) showed hybridization signals in centromeric region of all chromosomes in the studied species, although the intensity of the signals drastically differed. In <i>Th</i>. <i>intermedium</i>, the strongest signal of RT-CR probe was detected on the chromosomes of J^v, intermediate on J^r and faint on J^s and St subgenome suggesting different abundance of RT-CR on the individual chromosomes rather than the sequence specificity of RT-CRs of the subgenomes. RT-CR quantification using real-time PCR revealed that its content per genome in <i>Th</i>. <i>bessarabicum</i> is ~ 2 times and <i>P</i>. <i>spicata</i> is ~ 1,5 times higher than in genome of <i>D</i>. <i>villosum</i>. The possible burst of Ty3/gypsy centromeric retrotransposon in <i>Th</i>. <i>intermedium</i> during allopolyploidization and its role in proper mitotic and meiotic chromosome behavior in a nascent allopolyploid is discussed.</p></div