Caryologia. International Journal of Cytology, Cytosystematics and Cytogenetics 74(3): 3-8, 2021 Firenze University Press www.fupress.com/caryologia ISSN 0008-7114 (print) | ISSN 2165-5391 (online) | DOI: 10.36253/caryologia-1134 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Citation: Ahmet L. Tek, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım (2021) Chromomycin A3 banding and chromosomal mapping of 45S and 5S ribosomal RNA genes in bottle gourd. Caryologia 74(3): 3-8. doi: 10.36253/ caryologia-1134 Received: November 12, 2020 Accepted: Semptember 24, 2021 Published: December 21, 2021 Copyright: © 2021 Ahmet L. Tek, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım. This is an open access, peer-reviewed article published by Firenze University Press (http://www.fupress.com/caryo- logia) and distributed under the terms of the Creative Commons Attribution License, which permits 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. ORCID ALT: 0000-0002-3292-5142 KK: 0000-0001-7928-3290 HY: 0000-0003-2143-9242 Chromomycin A3 banding and chromosomal mapping of 45S and 5S ribosomal RNA genes in bottle gourd Ahmet L. Tek*, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım Department of Agricultural Genetic Engineering, Ayhan Şahenk Faculty of Agricultural Sciences and Technologies, Niğde Ömer Halisdemir University, 51240, Niğde, Turkey *Corresponding author. E-mail: altek2@gmail.com Abstract. Ribosomal DNAs and various banding patterns are landmarks in molecular cytogenetics providing useful information for karyotyping and addressing individual chromosomes. Bottle gourd is the only cultivated species of the Lagenaria genus with high genetic diversity. After CMA3/DAPI fluorochrome banding we investigated the GC- and AT-rich regions in interphase nuclei of five different local accessions. Fluo- rescence in situ hybridization (FISH) was conducted to determine the number and location of 45S and 5S rDNAs in bottle gourd. Our results showed four strong CMA3 regions in interphase and on mitotic metaphase chromosomes. FISH revealed four strong signals of 45S rDNA at the termini of two metaphase chromosome pairs and terminal 5S rDNA signals at another pair of chromosomes. The presence of four pos- itive CMA3 bands colocalizes with four 45S rDNA signals in all bottle gourd acces- sions. Our results allow distinguishing two out of eleven chromosome pairs of bottle gourd. Keywords: bottle gourd, chromomycin A3, fluorescence in situ hybridization, local accessions, ribosomal DNA, 5S, 45S. INTRODUCTION Bottle gourd, Lagenaria siceraria, is a member of the Cucurbitaceae family, also known as calabash or white-flowered gourd. It is a diploid crop species with 2n = 2× = 22 chromosomes (Beevy and Kuriachan 1996). The genome size of this plant is approximately 365 Mbp (Achigan-Dako et al., 2008). DNA markers revealed that Chinese bottle gourd and Turkish bottle gourd accessions have a close phylogenetic relationship within other cucurbit species (Xu et al., 2011; Yildiz et al., 2015). These findings have implications for the preservation of bottle gourd genetic diversity and advanced marker- assisted breeding studies (Xu et al., 2011; Yildiz et al., 2015). The Lagenaria genus consists of six species: L. siceraria (cultivated form), L. sphaerica, L. rufa, L. breviflora, L. sphaerica, and L. guineensis. All six species are natu- rally found in Africa, the supposed center of genetic diversity for L. siceraria. 4 Ahmet L. Tek, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım Bottle gourd is the only cultivated species. In the trop- ics, it is one of the oldest crops (Erickson et al., 2005). Bottle gourd is cultivated for food, decoration, medicine, domestic utensils, musical instruments, containers, and fishing floats (Morimoto and Mvere, 2004, Xu et al., 2011). Some bottle gourd varieties are grown for their seeds which are rich in oil and essential amino acids (Achigan-Dako et al., 2008). Additionally, bottle gourd seedlings are used as a rootstock for watermelon against adverse effects in soil such as low temperature, high pH, salinity, excessive water as well as soil-borne diseases, such as Fusarium wilt (Yetisir et al., 2007). For seedless watermelon production, bottle gourd pollen has recently been utilized to pollinate watermelons (Sugiyama et al., 2014). Therefore, bottle gourd is a crop of great economic interest and a detailed characterization at the chromo- some and genome level is desirable. The plant genomes contain a significant amount of repetitive DNA sequences. Among them, ribosomal DNAs (rDNAs) encode the RNA components of ribo- somes. Two structurally distinct gene families of rDNAs exist in plant genomes, specifically known as 45S and 5S rDNAs. Non-transcribed spacers and tandem repeat units of the 18S-5.8S-26S ribosomal genes are present in the 45S rDNA. The 5S rDNA genes consist of a non- transcribed spacer and a conserved coding region of 120 bp (Long and Dawid, 1980). 45S and 5S rDNA genes can be present at one or more positions within a set of chro- mosomes and be used as chromosomal markers (Long and Dawid, 1980; Lombello and Pinto-Maglio, 2007; Han et al., 2008; Heslop-Harrison and Schwarzacher, 2011; Li et al., 2016; Santos-Sanchês et al., 2019). CMA3 (chromomycin A3), a GC-rich specific f luorochrome, and DAPI (4’-6-diamidino-2-phenylindole), an AT-rich specific fluorochrome, banding techniques can also be useful to differentiate between chromosomes (Kim et al. 2002). The bottle gourd and its relatives have small and morphologically similar chromosomes, and fluo- rescent chromosome staining techniques such as CMA3 and DAPI might be helpful to distinguish them and proved to be useful for determining the phylogenetic relationships among plant species (Schweizer, 1976; Kim et al. 2002; Yamamoto et al., 2007; Volkov et al., 2017; Maragheh et al., 2019). Fluorescence in situ hybridiza- tion (FISH) has been widely used for constructing chro- mosomal maps, for chromosome identification, for stud- ying the dynamic organization of chromatin in inter- phase nuclei as well as for studying chromosome homol- ogy and karyotype evolution (Lysak et al., 2006; Probst, 2018; Santos et al., 2020). Previously, rDNA mapping in bottle gourd was investigated by Waminal and Kim (2012) and Li et al. (2016), although definite information is not available about origin of accessions used for comparison. These authors found four signals of 45S and two signals of 5S on metaphase chromosomes. There is no report for the CMA3/DAPI staining of the metaphase chromosome of bottle gourd. We hypothesize that a combination of CMA3/DAPI staining and FISH can be used for the determination of any possible chromosomal variabil- ity from different geographical origins. Given the high genetic diversity of bottle gourds, we tested a set of local varieties from Turkey for potential variability of rDNAs loci and differentiation of their metaphase chromosomes by CMA3/DAPI staining. MATERIALS AND METHODS Plant Material Bottle gourd (Lagenaria siceraria) seeds were obtained from a local population in Sandıklı, Afyon- karahisar, Turkey and used as a main accession for the research if not indicated otherwise. Additionally, four different accessions were used from provinces of Hatay, Niğde/Merkez, Niğde/Ulukışla, and Niğde/Bor (Turkey). These accessions with different seed morphology and high germination rates were chosen to cover different localities to determine variability. Moistened seeds were placed on sterile filter paper and germinated in petri dishes using double distilled water. Mitotic chromosome preparation The mitotic chromosome preparation was performed according to the published protocols (Tek et al. 2011) with the following modifications. Young root (~1 cm) tips were cut off with a razor blade, treated with 2 mM 8-hydroxyquinoline at room temperature, and subse- quently, fixed in 3:1 methanol: glacial acetic acid for 24 h at -20 °C. After fixation, root tips were washed three times in distilled water and 30 mM potassium chloride, digested in an enzyme mixture, pH 4.5, containing 4% cellulase and 2% pectinase at 37 °C for 90 min. Digested root tips were washed in distilled water. Following a fix- ation step, slides were prepared by the flame-dry (Tek et al. 2011). Slides with suitable cells were selected using a phase-contrast microscope. Chromomycin A3/DAPI staining Chromosome staining with CMA3 and DAPI was performed as described (Schweizer 1976). Briefly, slides 5Mapping ribosomal genes in bottle gourd were stained for 20 min with CMA3 (0.5 mg/ml) in Mcllvaine’s buffer (pH = 7.0). Subsequently, slides were incubated at 37 ºC for 2 days with DAPI (0.5 µg/ml) (Hasterok et al., 2006). Probe preparation To determine 45S and 5S rDNA sites, plasmid clones pTa71, and pTa794 were used, respectively (Gerlach and Bedbrook, 1979; Gerlach and Dyer 1980). Both rDNAs were labeled with digoxigenin-11-dUTP using a nick translation kit (Roche) according to the manufacturer’s instructions. Fluorescence in situ hybridization and signal detection FISH was conducted according to Tek et al., (2011) with modifications. A hybridization mixture containing the denatured probe DNA in 50% formamide, 10% dex- tran sulfate, 2×SSC was applied. The slides with chromo- somes were denatured in 70% formamide with 2×SSC at 80 °C for 2 min before hybridization at 37 °C overnight. The rhodamine-conjugated anti-digoxigenin antibody (Roche) was used to detect both 45S and 5S signals in independent experiments. Slides were checked using a fluorescence microscope at 63× magnification (Carl Zeiss Axio Imager.A2). Photographs were captured with mon- ochromatic charge-coupled device (CCD) camera (Carl Zeiss Axiocam 702) operated with multichannel ZEN Pro Imaging software. RESULTS AND DISCUSSION Chromosomes of a local bottle gourd accession were examined for the presence of GC- and AT-rich hetero- chromatin regions by CMA3/DAPI and by FISH for dis- tribution of rDNAs. GC-rich heterochromatin regions, as displayed by fluorochrome banding, proved to be cor- related with rDNA genes. In the interphase stage, four strong positive CMA3 bands were observed (Fig. 1a-c). Four strong positive CMA3 bands were also detected on the metaphase chromosomes (Fig. 1d-f ). Among these positive CMA3 bands were clear differences observed on the size and brightness on the interphase as well as the mitotic metaphase chromosomes (Fig. 1h, k, n, r). Two of the bands are small and less bright, while the other two are large and bright. These observations are consistent among all five accessions analysed indicating the simi- larity of the structural CMA3 bands. On the metaphase chromosomes, all four bright and strong CMA3 bands were present in termini regions of two pairs of chromo- somes. Also FISH with the 45S rDNA probe yielded four signals. In plants, 45S rDNA loci and CMA3 positive heterochromatic blocks often coincide spatially (Lombel- lo and Pinto-Maglio, 2007; Maragheh et al., 2019; San- tos-Sanchês et al., 2019). 5S rDNA sites were not detect- ed by CMA3/DAPI banding. The same number and posi- tion of ribosomal gene (45S rDNA) showed that CMA3/ DAPI positive bands may overlap with 45 rDNA sites. Similarly, our method, first with FISH experiment and subsequently with CMA3/DAPI banding procedure on the same interphase nuclei, does allow conclusive evi- dence of overlap between 45S rDNA signals and CMA3/ DAPI positive bands (Fig. 2a-c, d-f ). When we investi- gate CMA3 bands in terms of size and intensity, a pair of chromosomes have big and another pair of chromo- somes consistently have small signals in both metaphase and interphase stages whereas we did not detect clear differences on 45S rDNA signals using FISH (Fig. 2a-c, d-f ). Nevertheless, these differences are more prominent as presented in Fig. 1 Lombello and Pinto-Maglio, (2007) worked on the bitter gourd (Momordica charantia), which is a member of the same family as bottle gourd. They found no band on the chromosomes with DAPI staining. Santos-Sanchês et al., (2019) conducted similar work on different melon accessions and reported a DAPI positive band on the metaphase chromosomes. Chromo- somes stained with CMA3 revealed four bands in termi- nal regions of chromosomes in this species. Our results of CMA3/DAPI staining are in line with those obtained by Lombello and Pinto-Maglio, (2007) and Santos- Sanchês et al., (2019). To detect the position and number of 45S and 5S rDNAs, FISH with digoxigenin-labeled probes was applied. In interphase nuclei, four strong red signals of 45S rDNA were observed (Fig. 2a-c). The size and inten- sity of all four red signals were similar, which is in con- trast to our findings from the CMA3 bands. Four strong 45S rDNA signals were also detected on the short arm ends of two mitotic metaphase chromosome pairs (Fig. 2g-i). Two strong signals of 5S rDNA were observed in interphase nuclei (Fig. 2j-l) as well as on metaphase chromosomes (Fig. 2m-o). There was no prominent dif- ference in the size and intensity of 45S and 5S rDNA sig- nals. The 5S rDNA signals appeared on the short arm of termini of a metaphase chromosome pair (Fig. 2o). The two rDNA families are usually not positioned on the same chromosomes, with some exceptions (Waminal and Kim, 2012; Li et al., 2016). In conclusion, rDNA loci and CMA3 bands in accessions of Lagenaria siceraria, as in other species provide useful markers to distinguish at least two chromosome pairs individually. Also, our data 6 Ahmet L. Tek, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım demonstrate that a relatively low level of intraspecific chromosomal diversity is present among morphologi- cally different bottle gourd accessions. AUTHOR CONTRIBUTIONS ALT conceived the study and designed the experi- ments. HY, KK and ALT performed the experiments. HY, KK, BŞY and ALT conducted data analysis. HY, KK, BŞY and ALT wrote the paper. All authors read and approved the final manuscript. Figure 1. Chromomycin A3/DAPI (CMA3/DAPI) staining in Lagenaria siceraria nuclei and mitotic chromosomes (2n = 2× = 22) from five different local accessions. Interphase nuclei (a-c, g-i, j-l, m-o, p-s), prometaphase chromosomes (d-f ), GC-rich loci stained with CMA3 (b, e, h, k, n, r; green signals) and DAPI merge image (c, f, i, l, o, s) are shown on the chromosomes. Images are shown from the local accessions obtained from provinces of Sandıklı (a-f ), Hatay (g-i), Niğde/Ulukışla (j-l), Niğde/Merkez (m-o), Niğde/Bor (p-s). Scale bar = 5 μm. Figure 2. Localization of 45S and 5S rDNA in Lagenaria siceraria nuclei and mitotic chromosomes (2n = 2× = 22). DAPI stained interphase chromosomes (a-c), metaphase chromosomes (g-i), 45S rDNA loci labeled with digoxigenin (b, h; red signals of rho- damine), 45S rDNA loci merge image (c, i) is shown on the chro- mosomes. Interphase nuclei (d-f ), GC-rich loci staining with CMA3 (e; green signal). DAPI stained interphase chromosomes (j-l), meta- phase chromosomes (m-o), 5S rDNA loci labeled with digoxigenin (k, n; red signals of rhodamine), 5S rDNA loci merge image (l, o) is shown on the chromosomes. Scale bar = 5 μm. 7Mapping ribosomal genes in bottle gourd FUNDING T his work was pa r t ia l ly suppor ted by t he INDEPTH-COST Action CA16212 and scholarship pro- gram from Ayhan Şahenk Foundation. HY and BŞY rec- ognize the scholarship from YÖK 100/2000 Plant Genet- ics and Agricultural Biotechnology. ACKNOWLEDGMENTS We thank Prof. Ingo Schubert for critical reading and valuable suggestions on the manuscript and the lab members for technical assistance. REFERENCES Achigan-Dako EG, Fuchs J, Ahanchede A, Blattner FR (2008) Flow cytometric analysis in Lagenaria sicer- aria (Cucurbitaceae) indicates correlation of genome size with usage types and growing elevation. Plant Syst Evol 276:9–19. https://doi.org/10.1007/s00606- 008-0075-2 Beevy SS, Kuriachan P (1996) Chromosome numbers of south Indian Cucurbitaceae and a note on the cyto- logical evolution in the family. J Cytol Genet 31:65– 71 Erickson DL, Smith BD, Clarke AC, et al (2005) An Asian origin for a 10,000-year-old domesticated plant in the Americas. Proc Natl Acad Sci USA 102:18315– 18320. https://doi.org/10.1073/pnas.0509279102 Gerlach WL, Bedbrook JR (1979) Cloning and charac- terization of ribosomal RNA genes from wheat and barley. Nucleic Acids Res 7:1869–1885. https://doi. org/10.1093/nar/7.7.1869 Gerlach WL, Dyer TA (1980) Sequence organization of the repeating units in the nucleus of wheat which contain 5S rRNA genes. Nucleic Acids Res 8:4851– 4865. https://doi.org/10.1093/nar/8.21.4851 Han YH, Zhang ZH, Liu JH, et al (2008) Distribution of the tandem repeat sequences and karyotyping in cucumber (Cucumis sativus L.) by fluorescence in situ hybridization. Cytogenet Genome Res 122:80–88. https://doi.org/10.1159/000151320 Hasterok R, Wolny E, Hosiawa M, et al (2006) Compara- tive analysis of rDNA distribution in chromosomes of various species of Brassicaceae. Ann Bot 97:205– 216. https://doi.org/10.1093/aob/mcj031 Heslop-Harrison JSP, Schwarzacher T (2011) Organisation of the plant genome in chromosomes. Plant J 66:18– 33. https://doi.org/10.1111/j.1365-313X.2011.04544.x Kim ES, Punina EO, Rodionov AV (2002) Chromosome CPD(PI/DAPI)- and CMA/DAPI-banding patterns in Allium cepa L. Genetika 38:489–496 https://doi. org/10.1023/A:1015250219322 Li K-P, Wu Y-X, Zhao H, et al (2016) Cytogenetic rela- tionships among Citrullus species in comparison with some genera of the tribe Benincaseae (Cucurbi- taceae) as inferred from rDNA distribution patterns. BMC Evol Biol 16:85. https://doi.org/10.1186/s12862- 016-0656-6 Lombello RA, Pinto-Maglio CAF (2007) Cytomolecular studies in Momordica charantia L. (Cucurbitaceae), a potential medicinal plant. Cytologia 72:415–418. htt- ps://doi.org/10.1508/cytologia.72.415 Long EO and Dawid IB (1980) Repeated genes in eukary- otes. Annual Review of Biochemistry 49:727–764. https://doi.org/10.1146/annurev.bi.49.070180.003455 Lysak, M. A., Berr, A., Pecinka, A., Schmidt, R., McBreen, K., & Schubert, I. (2006). Mechanisms of chromo- some number reduction in Arabidopsis thaliana and related Brassicaceae species. Proceedings of the National Academy of Sciences, 103(13), 5224–5229. https://doi.org/10.1073/pnas.0510791103 Maragheh FP, Janus D, Senderowicz M, et al (2019) Kar- yotype analysis of eight cultivated Allium species. J Appl Genet 60:1–11. https://doi.org/10.1007/s13353- 018-0474-1 Morimoto Y, Mvere B (2004) Lagenaria siceraria. Back- huys Publishers/CTA, Wageningen/Leiden Probst AV (2018) A compendium of methods to analyze the spatial organization of plant chromatin. Methods Mol Biol 1675:397–418. https://doi.org/10.1007/978- 1-4939-7318-7_23 Santos AP, Gaudin V, Mozgová I, et al (2020) Tiding- up the plant nuclear space: domains, function and dynamics. J Exp Bot. https://doi.org/10.1093/jxb/ eraa282 Santos-Sanchês, R. de C. V., Souza, M. M., Melo, C. A. F. de, Silva, G. S., Xavier, R. C., Nunes, G. H. de S., & Araújo, I. S. (2019). Karyotypic characterization of melon accessions. Científica, 47(1), 91-103. https:// doi.org/10.15361/1984-5529.2019v47n1p91-103 Schweizer D (1976) Reverse fluorescent chromosome banding with chromomycin and DAPI. Chromosoma 58:307–324. https://doi.org/10.1007/BF00292840 Sugiyama K, Kami D, Muro T (2014) Induction of par- thenocarpic fruit set in watermelon by pollina- tion with bottle gourd (Lagenaria siceraria (Molina) Standl.) pollen. Scientia Horticulturae 171:1–5. htt- ps://doi.org/10.1016/j.scienta.2014.03.008 Tek AL, Kashihara K, Murata M, Nagaki K (2011) Func- tional centromeres in Astragalus sinicus include a 8 Ahmet L. Tek, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım compact centromere-specific histone H3 and a 20-bp tandem repeat. Chromosome Res 19:969–978. htt- ps://doi.org/10.1007/s10577-011-9247-y Waminal NE, Kim HH (2012) Dual-color FISH karyo- type and rDNA distribution analyses on four Cucur- bitaceae species. Hortic Environ Biotechnol 53:49–56. https://doi.org/10.1007/s13580-012-0105-4 Xu P, Wu X, Luo J, et al (2011) Partial sequencing of the bottle gourd genome reveals markers useful for phy- logenetic analysis and breeding. BMC Genomics 12:467. https://doi.org/10.1186/1471-2164-12-467 Volkov RA, Panchuk II, Borisjuk NV, et al (2017) Evo- lutional dynamics of 45S and 5S ribosomal DNA in ancient allohexaploid Atropa belladonna. BMC Plant Biol 17:21. https://doi.org/10.1186/s12870-017-0978-6 Yamamoto M, Abkenar AA, Matsumoto R, et al (2007) CMA banding patterns of chromosomes in major Citrus species. J Japan Soc Hort Sci 76:36–40. https:// doi.org/10.2503/jjshs.76.36 Yetişir, H., Kurt, Ş., Sari, N., & Tok, F. M. (2007) Root- stock potential of Turkish Lagenaria siceraria germ- plasm for watermelon: plant growth, graft compat- ibility, and resistance to Fusarium. Turk J Agric For, 31(6), 381-388. https://doi.org/10.3906/tar-0707-56 Yildiz M, Cuevas HE, Sensoy S, et al (2015) Transfer- ability of Cucurbita SSR markers for genetic diver- sity assessment of Turkish bottle gourd (Lagenaria siceraria) genetic resources. Biochemical Systemat- ics and Ecology 59:45–53. https://doi.org/10.1016/j. bse.2015.01.006 Caryologia International Journal of Cytology, Cytosystematics and Cytogenetics Volume 74, Issue 3 - 2021 Firenze University Press Chromomycin A3 banding and chromosomal mapping of 45S and 5S ribosomal RNA genes in bottle gourd Ahmet L. Tek*, Hümeyra Yıldız, Kamran Khan, Bilge Ş. Yıldırım Development of a protocol for genetic transformation of Malus spp Federico Martinelli1,*, Anna Perrone2, Abhaya M. 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