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

A Pipeline NanoTRF as a New Tool for De Novo Satellite DNA Identification in the Raw Nanopore Sequencing Reads of Plant Genomes

High-copy tandemly organized repeats (TRs), or satellite DNA, is an important but still enigmatic component of eukaryotic genomes. TRs comprise arrays of multi-copy and highly similar tandem repeats, which makes the elucidation of TRs a very challenging task. Oxford Nanopore sequencing data provide a valuable source of information on TR organization at the single molecule level. However, bioinformatics tools for de novo identification of TRs in raw Nanopore data have not been reported so far. We developed NanoTRF, a new python pipeline for TR repeat identification, characterization and consensus monomer sequence assembly. This new pipeline requires only a raw Nanopore read file from low-depth (<1×) genome sequencing. The program generates an informative html report and figures on TR genome abundance, monomer sequence and monomer length. In addition, NanoTRF performs annotation of transposable elements (TEs) sequences within or near satDNA arrays, and the information can be used to elucidate how TR–TE co-evolve in the genome. Moreover, we validated by FISH that the NanoTRF report is useful for the evaluation of TR chromosome organization—clustered or dispersed. Our findings showed that NanoTRF is a robust method for the de novo identification of satellite repeats in raw Nanopore data without prior read assembly. The obtained sequences can be used in many downstream analyses including genome assembly assistance and gap estimation, chromosome mapping and cytogenetic marker development

Molecular cytogenetic characterization of the dioecious Cannabis sativa with an XY chromosome sex determination system.

Hemp (Cannabis sativa L.) was karyotyped using by DAPI/C-banding staining to provide chromosome measurements, and by fluorescence in situ hybridization with probes for 45 rDNA (pTa71), 5S rDNA (pCT4.2), a subtelomeric repeat (CS-1) and the Arabidopsis telomere probes. The karyotype has 18 autosomes plus a sex chromosome pair (XX in female and XY in male plants). The autosomes are difficult to distinguish morphologically, but three pairs could be distinguished using the probes. The Y chromosome is larger than the autosomes, and carries a fully heterochromatic DAPI positive arm and CS-1 repeats only on the less intensely DAPI-stained, euchromatic arm. The X is the largest chromosome of all, and carries CS-1 subtelomeric repeats on both arms. The meiotic configuration of the sex bivalent locates a pseudoautosomal region of the Y chromosome at the end of the euchromatic CS-1-carrying arm. Our molecular cytogenetic study of the C. sativa sex chromosomes is a starting point for helping to make C. sativa a promising model to study sex chromosome evolution

The meiotic chromosomes of <i>C. sativa</i> at diakinesis (a) and metaphase I (b).

<p>The chiasma between the sex chromosomes can clearly be seen and indicated by arrows. <b>c</b> Idiogram of the <i>C. sativa</i> XY chromosomes with the hybridization sites of CS-1 (green) and the <i>Arabidopsis-</i>type telomeric repeat (red). The pseudoautosomal region is indicated by brackets.</p

Chromosomes of <i>C. sativa</i>: C-banding/DAPI, male metaphase (a), male prometaphase (b) and female metaphase (c).

<p>Distribution of <i>Arabidopsis-</i>type (5′-CCCTAAA-3′) telomeric sequences (small red dots located at the end of the chromosomes) (d). FISH with 45S rDNA (green signals) on male (e) and female (f) metaphase chromosomes. Bicolor FISH to male metaphase (g) with CS-1 subtelomeric repeat (red) and 5S rDNA probe (green). FISH to female metaphase (h) with CS-1 subtelomeric repeat (red). The karyotypes of male (i) and female (j) plants. Bar  =  5 µm.</p

Simplified phylogeny of Cannabaceae genus included in idiograms.

<p>The phylogeny is according to the reference of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Yang1" target="_blank">[59]</a>. Idiograms created based on data obtained in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Karlov1" target="_blank">[21]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-GrabowskaJoachimiak2" target="_blank">[23]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Divashuk1" target="_blank">[24]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Alexandrov1" target="_blank">[26]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Kim1" target="_blank">[55]</a> and in this study. 5S rDNA: green signals; 45S rDNA: red signals; species-specific subtelomeric repeats (HSR-1for <i>H. lupulus</i>, HJSR for <i>H. japonicus</i> and CS-1 for <i>C. sativa</i>): green signal. The position of pseudoautosomal region on sex chromosomes is indicated by brackets. Time of divergence estimated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Murakami1" target="_blank">[60]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Murakami2" target="_blank">[61]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0085118#pone.0085118-Zerega1" target="_blank">[62]</a>.</p

Size of sex chromosomes compared to the largest autosome.

<p>Size of sex chromosomes compared to the largest autosome.</p

Idiogram of haploid chromosome complement of <i>C. sativa,</i> including <i>Arabidopsis-</i>type telomeric repeat, CS-1 subtelomeric repeat, 45S rDNA and 5S rDNA sites.

<p>Idiogram of haploid chromosome complement of <i>C. sativa,</i> including <i>Arabidopsis-</i>type telomeric repeat, CS-1 subtelomeric repeat, 45S rDNA and 5S rDNA sites.</p

Characterization of repeated DNA sequences in genomes of blue-flowered flax

Abstract Background Members of different sections of the genus Linum are characterized by wide variability in size, morphology and number of chromosomes in karyotypes. Since such variability is determined mainly by the amount and composition of repeated sequences, we conducted a comparative study of the repeatomes of species from four sections forming a clade of blue-flowered flax. Based on the results of high-throughput genome sequencing performed in this study as well as available WGS data, bioinformatic analyses of repeated sequences from 12 flax samples were carried out using a graph-based clustering method. Results It was found that the genomes of closely related species, which have a similar karyotype structure, are also similar in the repeatome composition. In contrast, the repeatomes of karyologically distinct species differed significantly, and no similar tandem-organized repeats have been identified in their genomes. At the same time, many common mobile element families have been identified in genomes of all species, among them, Athila Ty3/gypsy LTR retrotransposon was the most abundant. The 30-chromosome members of the sect. Linum (including the cultivated species L. usitatissimum) differed significantly from other studied species by a great number of satellite DNA families as well as their relative content in genomes. Conclusions The evolution of studied flax species was accompanied by waves of amplification of satellite DNAs and LTR retrotransposons. The observed inverse correlation between the total contents of dispersed repeats and satellite DNAs allowed to suggest a relationship between both classes of repeating sequences. Significant interspecific differences in satellite DNA sets indicated a high rate of evolution of this genomic fraction. The phylogenetic relationships between the investigated flax species, obtained by comparison of the repeatomes, agreed with the results of previous molecular phylogenetic studies