Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 73(3): 121-126, 2020 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.13128/caryologia-578 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: J.M. Rodríguez-Domínguez, E. Tapia-Campos, R. Barba-Gonzalez (2020) Physical mapping of 45S and 5S rDNA in two Sprekelia formosissima cytotypes (Amaryllidaceae) through Fluorescent In Situ Hybridization (FISH). Caryologia 73(3): 121-126. doi: 10.13128/caryologia-578 Received: July 31, 2019 Accepted: July 14, 2020 Published: December 31, 2020 Copyright: © 2020 J.M. Rodríguez- Domínguez, E. Tapia-Campos, R. Bar- ba-Gonzalez. This is an open access, peer-reviewed article published by Firenze University Press (http://www. fupress.com/caryologia) and distributed under the terms of the Creative Com- mons Attribution License, which per- mits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All rel- evant data are within the paper and its Supporting Information files. Competing Interests: The Author(s) declare(s) no conflict of interest. Physical mapping of 45S and 5S rDNA in two Sprekelia formosissima cytotypes (Amaryllidaceae) through Fluorescent In Situ Hybridization (FISH) José Manuel Rodríguez-Domínguez, Ernesto Tapia-Campos, Rodrigo Barba-Gonzalez* Unidad de Biotecnología Vegetal, Centro de Investigación y Asistencia en Tecnología y Diseño del Estado de Jalisco, Guadalajara, Jalisco, México *Corresponding author. E-mail: rbarba@ciatej.mx Abstract. Chromosome number and position of rDNA were studied in plants of Sprekelia formosissima (Amaryllidaceae) collected in two populations with different ploidy level (2n=2x=60 and 2n=5x=150). The 5S and 45S rRNA loci were localized and physically mapped using two-color fluorescence in situ hybridization probes. The diploid (2n=2x=60) cytotype showed four loci for the 45S rDNA in two chromosome pairs (11 and 25) in telomeric position. The 5S rDNA was present in six loci of three homologous chromosome pairs (3, 13 and 19) in subtelomeric and telomeric positions. The chromosomes of the pentaploid cytotype (2n=5x=150) showed five loci for the 45S rDNA in telomeric position and five loci for the 5S rDNA in subtelomeric position. The karyotypic formula is 13m + 16sm + 1 st and the karyotype symmetry/asymme- try index is TF % = 34.67, AsK % = 65.32 and Syi % = 54.81, concluding that it is an asymmetric karyotype, bimodal with one distinctively large pair of chromosomes (10.42 µm) and a gradual decrease in the size of the other chromosome pairs, from the longest of 6.84 µm, to the shortest of 2.61 µm. Keywords: bulbous genus, karyogram, karyotypic formula, ornamental, plant chro- mosomes, ploidy level. INTRODUCTION Sprekelia Heist, is a bulbous genus of the monocotyledonous family Amaryllidaceae, it is a perennial, herbaceous monotypic genus commonly known as Aztec lily or Jacobean lily represented by the sole species Spreke- lia formosissima (Flory 1977; Sánchez 1979); it is distributed through Mexico and Guatemala (López-Ferrari and Espejo-Serna 2002). Its scarlet flowers with curved petals have made of it an exceptional ornamental pot plant. The technique of Fluorescent In Situ hybridization (FISH) has become a very useful tool for the detection of specific DNA in the genome of organ- isms. In this technique, the genes encoding the 5S ribosomal RNA (rDNA 122 José Manuel Rodríguez-Domínguez, Ernesto Tapia-Campos, Rodrigo Barba-Gonzalez 5S) and the 18S-5.8S-26S ribosomal RNA (45S rDNA) are commonly used as markers for the physical mapping of plant chromosomes due to the high number of their repeating units, specific position on chromosomes and highly conserved sequences (Liu and Davis 2011). The 45S ribosomal RNA genes are clustered in tandem arrays of repeating units of the genes 18S, 5.8S and 26S, inter- nal transcribed spacer sequences (ITS) and external non- transcribed spacer sequences (NTS), with an approxi- mate size of 7.5-18.5 Kb in plants (Mizuochi et al. 2007). The 5S ribosomal RNA genes are also found in tandem repeats with an approximate size of 0.2-0.9 Kb, with a highly conserved region (120 bp long) separated by a NTS sequence (Specht et al. 1997). Physical mapping of rDNA has been performed in many plants such as: maize (Li and Arumuganathan 2001), orchids (Cabral et al. 2006), Crotalaria juncea (Mondin et al. 2007), Agave (Robert et al. 2008; Gomez-Rodriguez et al. 2013), aspar- agus (Deng et al. 2012), Lilium (Lim et al. 2001; Hwang et al. 2015), Tigridia pavonia (Arroyo-Martínez et al. 2018), among many others, however, there is little work in plants of the Amaryllidaceae family using these mod- ern techniques of molecular cytogenetics. The objective of this study was the physical mapping of rDNA 45S and 5S in two cytotypes (diploid and pentaploid) of Sprekelia formosissima. MATERIAL AND METHODS Plant material Sprekelia formosissima bulbs were collected from two different populations and maintained in an in vivo collection at the Centro de Investigación y Asisten- cia en Tecnología y Diseño del Estado de Jalisco A.C. (CIATEJ). Two cytotypes were found, a diploid popu- lation (2n=2x=60) from the road Villa-Corona-Cocula, and a pentaploid population (2n=5x=150) from a pri- vate collection in Zapopan, both in the state of Jalis- co, México. The bulbs were placed in a container with moist substrate composed by a mixture of Peat Moss and Vermiculite (7:3) for the bulbs to produce roots. Sampling was performed in ten bulbs of each of the two populations. Chromosome spreads Obtaining metaphasic chromosomes was per- formed as described by Rodríguez-Domínguez et al. (2017), in brief, the root apex were pretreated in a satu- rated solution of α-bromonaphthalene (0.1%) for 48 h at 4 °C and fixed in a methanol:acetic acid (3:1) solution for 24 h at 4 °C. Afterwards, the root apex were washed with milli-Q water, macerated in an enzyme mixture containing each 0.2% (w/v) of pectolyase Y23, RS cellu- lase and cytohelicase in 10 mM citrate buffer (pH 4.5) and incubated at 37 °C for 3 h. Later, a cell suspension was obtained by vortexing, followed by washing with distilled water, centrifuging at 10,000 rpm for 45 s, washed with methanol, then, centrifuged at 11,000 rpm during 30 s. The cell pellet was resuspended in 100 µl methanol. Finally, in a fume hood, the slides were cov- ered with a layer of pure acetic acid and 10 µl of cell suspension were added to each slide. The slides were immediately inclined at an angle of 45 degrees, and two drops of pure acetic acid were added; the slides were exposed (facing down) to vapor from a water bath at 55 °C for 5 s, finally, a drop of pure acetic acid was added and allowed to air dry. DNA probes Two different clones were utilized as probe, clones pTa71 and pTa794 which contains the EcoRI fragment of 45S and 5S ribosomal DNA from wheat respectively (Gerlach and Bedbrook 1979; Gerlach and Dyer 1980). The bacteria were cultured for 24 hours under constant agitation (225 rpm) at 37 °C in LB medium supple- mented with 0.1 mg/ml ampicillin. Isolation of wheat ribosomal DNA was performed using the kit High Pure Plasmid Isolation Kit (Roche®) according to the sup- plier’s instructions. In brief, 4 ml of the inoculated cul- ture medium were transferred to 15 ml tubes and cen- trifuged at 9,000 rpm for 1 minute, the supernatant was discarded; the bacterial pellet was resuspended in 250 µl of suspension buffer and transferred to a 1.5 ml tube. 250 µl of lysis buffer were added and mixed by inversion, then incubated for 5 min and 350 µl of binding buffer were added and incubated for 5 min on ice; followed by centrifugation at 13,000 rpm for 10 min. The superna- tant was transferred to a filtration membrane and cen- trifuged at 13,000 rpm for 1 minute; the membrane was transferred to a new tube and 500 µl of washing buffer I was added and centrifuged at 13,000 rpm for 1 minute. The column was transferred to a new tube and 700 µl of washing buffer II was added; centrifuged at 13,000 rpm for 1 minute. The flowthrough liquid was discarded and the column was centrifuged at 13,000 for 1 minute to dry the membrane, which was transferred to a new 1.5 ml tube and 100 µl of elution buffer was added and cen- trifuged at 13,000 rpm for 1 minute. 123Physical mapping of 45S and 5S rDNA in two Sprekelia formosissima cytotypes through FISH Labeling the probes Fluorescent in situ hybridization (FISH) was per- formed using two different probes (45S and 5S wheat rDNA) which were direct labelled as follows: 1 µg of rDNA in 12 µl mQ water, 4 µl Nick translation mix (Roche®) and 4 µl of 5x fluorophore mix (5 µl of each 2.5 mM dATP, dCTP, dGTP, 3.4 µl 2.5 mM dTTP, 4 µl 1 mM labeled dUTP and 27.6 µl mQ water) were incubat- ed at 15 °C for 90 min. Reaction was stopped by adding 1 µl of 0.5 M EDTA (pH 8.0) and heating at 65 °C for 10 min. 45S rDNA was labeled with Tetramethylrhoda- mine-5-dUTP and 5S rDNA with Fluorescein-12-dUTP. Fluorescent in situ hybridization Slides with metaphase chromosomes were incu- bated at 37 °C overnight, the next day, each slide was incubated in 200 μl RNase A (100 μg/ml) in 2xSSC at 37 °C for 1 h and washed three times for 5 min each in 2x SSC, incubated in 0.01 M HCl for 2 min. 200 μl Pepsin (5 μg/ml) were added and incubated at 37 °C for 10 min, washed in mQ water for 2 min and in 2x SSC for 5 min each. The slides were incubated in 4% para- formaldehyde for 10 min and washed three times in 2x SSC for 5 min each. The slides were dehydrated in 70%, 90% and absolute ethanol series for 3 min each and air- dried. Hybridization followed using a mixture consisting of 20x SSC, 50% formamide, 10% sodium dextran sul- phate, 10% SDS and 25-50 ng of each probe. The DNA was denatured by heating the hybridization mixture at 70 °C for 10 min and then placed on ice for at least 10 min. For each slide, 40 μl of hybridization mixture was used. The preparations were denatured at 80 °C for 10 min and incubated overnight in a humid chamber at 37 °C. After overnight hybridization, the slides were washed three times in 2x SSC at 37 °C for 5 min each, in the last wash temperature was increased to 42 °C and the slides were then washed three times in 0.1x SSC at 42 °C each followed by three washes in 2x SSC at RT for 5 min each. Chromosomes were counterstained with 1μg/ ml DAPI (4,6-diamidino-2-phenylindole) and a drop of Vectashield anti-fade (Vector laboratories) was added for its examination under a Leica DMRA2 microscope equipped with epi-fluorescent illumination, filter sets of DAPI, FITC and Cy3. Images were captured by a Evolu- tion QEi (Media Cybernetics) monochrome digital cam- era and processed with Image-Pro Plus software. The DAPI images were sharpened with a 7x7 High Gauss spatial filter. DAPI, FITC and Tetramethylrhodamine fluorescence were pseudo-colored with their respective dye tint for the FISH analyses. Karyotype analysis Karyotype analysis was based on at least 10 high- quality metaphase plates with almost similar degree of chromatin condensation, chromosome measurements were made using the freeware software Micromeasure (Reeves 2001) vers 3.3. The following measurements of each pair of chromosomes were made: Chromosome length, long arm (L), short arm (S), arm ratio (L/S). The chromosomes were assorted into different categories based on arm’s ratio arranged according to Levan et al. (1964) and the karyogram was performed; the chromo- some homology was assigned according to similarities in length, morphology and centromere position. The num- ber of homologous chromosomes was assigned sequen- tially according to the reduction of the chromosomal length and in base to centromere position. Three different methods of evaluating karyotype asymmetry were used: the TF% (Huziwara, 1962), the AsK% (Arano, 1963) and the Syi (Greilhuber and Speta, 1976; Venora et al. 2002). RESULTS The physical distribution of the 45S and 5S rDNA were investigated by fluorescent in situ hybridization (FISH) (Figures 1 to 3) in two cytotypes of Sprekelia formosissima, a diploid (2n=2x=60) and a pentaploid (2n=5x=150). The diploid (2n=2x=60) cytotype showed Figure 1. Fluorescent in situ hybridization of wheat 45S (red sig- nals, arrowheads) and 5S rDNA (green signals, arrows) in a diploid Sprekelia formosissima (2n=2x=60) cytotype. The chromosomes were counterstained with DAPI. Bar=10µm. 124 José Manuel Rodríguez-Domínguez, Ernesto Tapia-Campos, Rodrigo Barba-Gonzalez four loci for the 45S rDNA in two chromosome pairs (11 and 25) in telomeric position. The 5S rDNA was present in six loci of three homologous chromosome pairs (3, 13 and 19) in subtelomeric and telomeric positions (Figures 1 and 3). Chromosome pair 1 was identified due to its unmistakable large size. The chromosomes of the pen- taploid cytotypes (2n=5x=150) showed five loci for the 45S rDNA in telomeric position and five loci for the 5S rDNA in subtelomeric position (Figure 2). The hybridi- zation loci for each probe was found always in different chromosomes. Thirty chromosome pairs (60 chromo- somes in total) were identified in the diploid cytotype of S. formosissima and were arranged based on arm’s ratio according to Levan et al. (1964) in the karyogram shown in Figure 3. The karyotypic formula was 13m + 16sm + 1 st with predominance of m and sm chromosomes. The longest per shortest chromosome ratio (L/S) ranges from 1.00 to 5.12. The karyotype symmetry/asymmetry index is TF % = 34.67, AsK % = 65.32 and Syi % = 54.81, concluding that it is an asymmetric karyotype, bimodal Figure 2. Fluorescent in situ hybridization of wheat 45S (red sig- nals, arrowheads) and 5S rDNA (green signals, arrows) in a pen- taploid Sprekelia formosissima (2n=5x=150) cytotype. The chromo- somes were counterstained with DAPI. Bar=10µm. Figure 3. Karyogram of diploid Sprekelia formosissima. The wheat 45S rDNA hybridization loci (red signal) are present in chromo- some pairs 11 and 25, while the wheat 5S rDNA loci (green signal) are present in chromosome pairs 3, 13 and 19. Table 1. Chromosome parameters of Sprekelia formosissima Chromosome pair no. Total length (µm) Long arm (L) (µm) Short arm (S) (µm) Arm ratio L/S Centromeric position 1 10.42 5.40 5.02 1.08 M 2 5.02 3.10 1.92 1.61 M 3 3.98 2.29 1.69 1.36 M 4 3.86 2.22 1.64 1.35 M 5 3.60 1.96 1.64 1.20 M 6 3.41 1.95 1.46 1.34 M 7 3.26 1.71 1.55 1.10 M 8 3.24 1.89 1.35 1.40 M 9 3.19 1.81 1.38 1.31 M 10 3.14 1.89 1.25 1.51 M 11 3.06 1.55 1.51 1.03 M 12 2.87 1.57 1.30 1.21 M 13 2.61 1.36 1.25 1.09 M 14 6.84 4.77 2.07 2.30 Sm 15 6.46 4.70 1.76 2.67 Sm 16 6.15 4.25 1.90 2.24 Sm 17 6.08 4.46 1.62 2.75 Sm 18 6.04 4.3 1.74 2.47 Sm 19 5.50 4.12 1.38 2.99 Sm 20 5.43 4.07 1.36 2.99 Sm 21 5.22 3.77 1.45 2.60 Sm 22 5.16 3.63 1.53 2.37 Sm 23 5.14 3.63 1.51 2.40 Sm 24 5.04 3.32 1.72 1.93 Sm 25 4.91 3.19 1.72 1.85 Sm 26 4.61 3.15 1.46 2.16 Sm 27 4.31 2.92 1.39 2.10 Sm 28 4.16 2.68 1.48 1.81 Sm 29 3.68 2.50 1.18 2.12 Sm 30 6.84 5.41 1.43 3.78 St 125Physical mapping of 45S and 5S rDNA in two Sprekelia formosissima cytotypes through FISH with one distinctively large pair of chromosomes (10.42 µm) and a gradual decrease in the size of the other chro- mosome pairs, from the longest of 6.84 µm, to the short- est of 2.61 µm. (Table 1). DISCUSSION According to Roa and Guerra (2012), the most fre- quent loci number of 45S rDNA in angiosperms is two or four per diploid karyotype and they are generally located in terminal regions of the chromosomes, found more frequently in the short arms. In the cytotypes of S. formosissima analyzed in this work, both signals (45S and 5S rDNA) were detected in the short arms of the chromosomes. Even though the localization sites and the number of copies of the 5S and 45S loci present varia- tion, it is probably not related to the ploidy level as has been suggested in some studies (Weiss-Schneeweiss and Schneeweiss 2013). This coincides with our results where four 45S rDNA sites and six 5S rDNA sites were detected in diploid plants, while five 45S rDNA sites and five 5S rDNA sites were detected in the pentaploid cytotype. There may be a different number of signals in species that have the same ploidy level, for example, García (2015) reported four 45S sites and nine 5S sites for diploid Sprekelia howardii Lehmiller, while in our work only four 45S sites were detected and six 5S sites for diploids analyzed. Differences in signal sizes were also detected even when they were in homologous chro- mosomes, which may be due to the existence of differ- ent copy numbers of the rDNA genes in each chromo- some. There was a difference regarding the relation of the number of signals of rDNA in the cytotypes ana- lyzed in this study; in the diploid cytotype there was less 45S signals with respect to 5S signals, while a similar number of rDNA signals were detected in the pentaploid cytotype. Based on the chromosome number of the dip- loid cytotype (2n=60) it can be considered as a polyploid due to its high chromosome number (Stebbins 1971), with this in mind, the lower 45S rDNA loci could be explained since these are generally more prone to expe- rience rapid homogenization, silencing, and loss of loci, especially in polyploids (Clarkson et al. 2005; Kovařík et al. 2005; Weiss-Schneeweiss et al. 2008; Kotseruba et al. 2010). According the symmetry/asymmetry indexes used, the analyzes established that it is an asymmetric karyotype, however due to the predominance of meta- centric and submetacentric chromosomes, it was impos- sible to differentiate them only by their size, for this rea- son the number of individual chromosomes identified was relatively low. 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