The original code of the program was written in Java 8 using IntelliJ IDEA as the integrated development environment and is compatible with any Java-enable system with a runtime level of ≥1.7. Microsoft Excel version 10.0 or higher is required.

FISH Fluorescent in situ hybridization

PCR Polymerase chain reaction

GISH Genomic in situ hybridization

For cytogenetic experiments chromosomes were prepared using the SteamDrop method (Kirov et al. 2014). To visualise 5S and 45S rDNA in Allium fistulosum Linnaeus 1753, the plasmids pSct7 (Lawrence and Appels 1986) and pTa71 (Gerlach and Bedbrook 1979) were used. FISH was performed as described in Kirov et al. (2016). Biotin and digoxigenin labeled probes were detected by Streptavidin-Cy3 (Sigma-Aldrich, USA) and anti-digoxigenin-FITC (Roche, Germany), respectively.

GISH on × Festulolium Ascherson & Graebner, 1902 hybrids was performed as described in Van Laere et al (2010). Lolium perenne Linnaeus, 1753 was used as probe DNA, labelled with Digoxigenin, while Festuca pratensis Hudson, 1762 was used as block DNA.

Images were taken by a Zeiss AxioImager M1 fluorescence microscope (400× and 1000× magnification) equipped with an AxioCam MRm camera and ZEN software (Zeiss, Zaventem, Belgium).

DRAWID contains two main modules: (1) idiogram manipulations and (2) chromosome measurements (Figure 1A). DRAWID can draw an idiogram using data from the second module as well as from DRAWID generated Microsoft XL output tables. The first module provides an interface for idiogram manipulation, storage, analysis as well as for data representation. Chromosome order, name, and color and centromere color can be changed using the top menu and the pop-up menu after chromosome selection. Once idiogram adjustments are performed, the idiogram can be added to the DRAWID storage to use it later again. From this window high quality pictures can be saved in PNG format. Data from DRAWID can be saved as XL Microsoft files (.xlsx). The excel data files contain two sheets in total, one sheet with chromosome parameters such as chromosome length, centromere index (short arm length × 100 / total chromosome length), relative chromosome length, short arm and long arm lengths, and one metadata sheet containing information about signal and band positions and size. Building an idiogram for cytogenetically uncharacterized species requires an average idiogram based on measurements of several metaphases. To facilitate this process we implemented a function to build an average karyotype from multiple datasets stored in the DRAWID storage. After application of this function measurements of chromosomes with the same names will be used to calculate means for centromere index, whole chromosome length, short and long arm lengths and standard deviation. An average idiogram is plotted with error bars representing the standard deviation. This idiogram can again be exported and saved either as an image file or as a table. To build idiograms for haploids we inserted a function to merge neighboring (chromosome ordering is just performed by length) chromosomes depending on the ploidy level.

The second module (Figure 1A, B) allows chromosome measurements as well as chromosomal landmarking and includes a number of useful tools to simplify the measurement process. Chromosome and landmark names appear on the picture once the measurement is completed. FISH signal positions can be drawn on the idiogram. Correct positioning of the signals on the idiogram is becoming annoying when many probes and/or loci are involved. Therefore, DRAWID is equipped by a set of functions to correctly position all the signals on the idiogram and to easlily change their colors and names using the icons in the top panel menu. Chromosome name changes in the idiogram are automatically and immediately synchronized with the measurements on the picture. In addition, the visualized chromosome on the right side of the panel helps to identify the chromosomes and to link the idogram to the original picture. Sometimes, chromosomes have fragile sites and as a consequence, different fragments of these chromosomes can be positioned on different locations in the metaphase. To virtually join two or more fragments of the same chromosome and build an idiogram, we also implemented a specific function.

Figure 1.

Structure (A) and main windows of DRAWID (B, C). B Interface of chromosome measurement window, containing useful tools for chromosome and FISH/GISH signal measurements. The photograph in this panel shows the result of a FISH experiment on chromosomes of Allium fistulosum with biotin-labeled 5S rDNA and HAT58 repeat (Kirov et al. 2017). Lines and signatures show the path of chromosome and signal measuring C Interface for idiogram manipulation. This panel shows the idiogram of A. fistulosum constructed based on chromosome measurements and FISH (5S rDNA and HAT58 tandem repeat) signal positions in panel B Buttons at the top of the panel are used for chromosome and centromere color changing, display legend, chromosome order correction and idiogram storage manipulation. When a chromosome in the constructed idiogram is selected (entire chromosome 11 highlighted in red the image of the selected chromosome along with the parameters of its measurement appear on the screen (on the right of the panel C).

All measurements can be scaled by measuring the scale bar in the picture and using the scale bar button. As some errors can occur during measurements, DRAWID has several functions to remove measurements of certain chromosomes. Some frequently used functions such as marking a coordinate as a centromere or FISH signal/band, finishing chromosome measurements have both a hotkey and an icon.

The DRAWID program has a dedicated web page (http://www.drawid.xyz) on the website of the Department of plant genetics, biotechnology and breeding of Russian State Agrarian University – MTAA. All the described functions and some other functions are explained there in detail in the manual. In addition, version history and information about reported and solved bugs are published here and will be updated on a regular basis.

Example 1. Basic karyotyping and averaged idiogram

In order to assess DRAWID for karyotyping of individual metaphases (Figure 2A, B) as well as of a set of metaphases (Figure 2C, D), we used previously published data from karyotyping of Cannabis sativa Linnaeus, 1753 (Divashuk et al. 2014), Rosa wichurana Crépin, 1888 (Kirov et al. 2016), Allium cepa Linnaeus, 1753 and A. fistulosum (de Vries and Jongerius 1988, Kirov et al. 2017). All tested species are diploid, 2n = 2x = 20 (Cannabis sativa), 2n = 2x = 14 (Rosa wichurana), 2n = 2x = 16 (Allium cepa and A. fistulosum). Idiograms for these species constructed by DRAWID are presented in Figure 2. Our results fully coincided with the data of karyotyping published earlier.

The idiogram can also represent a monoploid chromosome set. For this, DRAWID has a function to convert a diploid or polyploid idiogram into a monoploid one and calculate the mean chromosome index and arm length of homologuous chromosomes. A new idiogram can then be drawn with indication of standard deviation bars. To demonstrate this, measurements of Allium fistulosum metaphase and idiogram construction using the function ‘reduce karyo’ was performed in DRAWID (Figure 2C).

Figure 2.

Examples of basic karyotype measurements and idiogram constuction by DRAWID. A Chromosomes of C. sativa (2n = 20); sex (black color) and NOR-bearing (green color) chromosomes are highlighted B Idiogram of Rosa wichurana; satellites on chromosomes 13 and 14 are colored in red C Idiogram of Allium fistulosum after measurement of the chromosomes, and application of the function “reduce karyo” merging homologuous chromosomes to obtain a monoploid idiogram, standard deviation bars are shown D Idiogram of Allium cepa constructed after measurements of 3 metaphases and application of the “get average karyo” function to obtain the average idiogram, standard deviation bars are shown.

In another example, three metaphases of Allium cepa were measured, data were collected into the storage container and an average idiogram was obtained using the “average karyo” function (Figure 2D). Standard deviations of chromosome arm lenghts and centromere indices are presented which provide useful information for the estimation of chromosome parameter variability and comparative analysis.

Example 2. FISH based idiogram

One major added value of DRAWID compared to other software is that it allows to measure FISH signals and indicate them on the idiogram. Figure 3 shows an idiogram construction of an A. cepa metaphase with HAT58 and CAT36 signals. The position of the signals and size (in case of bands) were calculated. Based on these measurements, the idiogram was build by DRAWID, using the scale bar for calibration, and signal positions of HAT58 and CAT36 rDNA probes were indicated.

Figure 3.

A Metaphase chromosomes of A. fistulosum after FISH with HAT58 (green signal) and CAT36 (red signals) repeats. Numbers in brackets correspond to the numbers on the idiogram (C) B The same picture as in A but after measurements by DRAWIDC Idiogram obtained from the measurements in A and B Scale bar – 10 µm.

Example 3. Idiogram after GISH experiments

GISH is a commonly used tool to study genome composition after interspecific crosses. A correctly drawn idiogram with indication of recombination points is important for result interpretation. We tested DRAWID to build idiograms from F2 hybrids between species of Lolium Linnaeus, 1753 and Festuca Linnaeus, 1753, having a complex genomic constitution with several recombination points. Using DRAWID, chromosome number, chromosome morphology and GISH signals were determined. On the idiogram, parental composition and sites of recombination are clearly visible (Figure 4).

Figure 4.

Idiogram (right) obtained from metaphase measurements of an F2 × Festulolium hybrid (2n = 34) after GISH analysis (left) with Lolium perenne genomic DNA labeled as a probe (Dig; green pseudocolor) and Festuca pratensis used as block DNA, counterstained with DAPI (blue pseudocolor). DRAWID measurements are shown for the recombinant chromosome 22 (arrow, CEN = centromere). Chromosome numbering is according to chromosome length starting with the largest chromosome.

IK, LK, KVL wrote the paper and designed the experiments. DR, AS, SM, IF tested and debugged the program. IK designed the webpage.