Genet Resour Crop Evol DOI 10.1007/s10722-013-9976-1 RESEARCH ARTICLE Cytomolecular characterization of rDNA distribution in various Citrullus species using fluorescent in situ hybridization Umesh K. Reddy • Nischit Aryal • Nurul Islam-Faridi • Yan R. Tomason Amnon Levi • Padma Nimmakayala • Received: 10 September 2012 / Accepted: 28 January 2013 Ó Springer Science+Business Media Dordrecht 2013 Abstract The 18S–28S and 5S rDNA sites are useful chromosome landmarks and provide valuable evidence about genome organization and evolution. This investigation was the first attempt to study the dynamics, distribution and directionality of rDNA gains and losses, as well as to understand the contribution of site number variation in the speciation of the genus Citrullus. In this study, we employed fluorescent in situ hybridization (FISH), using the18S– 28S and 5S rDNA gene loci, to evaluate the differences between the (1) cultivated type watermelon C. lanatus var. lanatus (sweet watermelon), (2) the ‘‘bitter’’ desert watermelon C. colocynthis (colocynth) that is indigenous to the deserts of northern Africa, the U. K. Reddy
N. Aryal
Y. R. Tomason
P. Nimmakayala (&) Department of Biology, Gus R. Douglass Institute, West Virginia State University, Institute, WV 25112-1000, USA e-mail: padma@wvstateu.edu U. K. Reddy e-mail: ureddy@wvstateu.edu N. Islam-Faridi U.S. Forest Service, Washington, DC, USA N. Islam-Faridi Department of Ecosystem Science and Management, Texas A&M University, College Station, TX 77843, USA A. Levi U.S. Vegetable Laboratory, USDA, ARS, 2875 Savannah Highway, Charleston, SC 29414, USA Middle East and Asia, (3) the C. lanatus var. citroides (citron) ‘‘Tsamma’’ or ‘‘cow watermelon’’ that is known as and is indigenous to southern Africa, (4) and C. rehmii that thrive in the Namibian Desert. The FISH analyses showed that the sweet watermelon and colocynth have similar rDNA configuration. The sweet watermelon and colocynth genomes contain two 18S–28S rDNA gene loci, each located on a different chromosome, and one 5S rDNA locus which is co-localized with one of the 18S–28S rDNA gene loci. On the other hand, the C. rehmii has one 18S–28S rDNA locus and one 5S rDNA locus positioned on different chromosomes, while the citron has one18S– 28S rDNA and two 5S rDNA loci, each located on a different chromosome. A FISH analysis of F1 (citron 9 sweet watermelon) chromosome spreads revealed uniparental homeologous rDNA gene copies pertaining to the sweet watermelon versus the citron chromosomes, with the sweet watermelon chromosome containing the 18S–28S and 5S rDNA locus versus the citron homologue chromosome that has the 5S rDNA locus, but not the 18S–28S rDNA locus. Genomic in situ hybridization (GISH) analysis, using the entire citron genome as a probe to be differentially hybridized on sweet watermelon chromosome spreads, revealed that the citron genomic probes mainly hybridize to subtelomeric and pericentromeric regions of the sweet watermelon chromosomes, suggesting extensive divergence between the citron and sweet watermelon genomes. The FISH and GISH cytogenetic analysis here indicate major differences in 123 Genet Resour Crop Evol genome organization between the cultivated watermelon type sweet watermelon and its counterpart citron that thrive in southern Africa and considered a useful germplasm source for enhancing disease and pest resistance in watermelon cultivars. Keywords Citrullus
FISH
rDNA organization
5S rDNA
18S–28S rDNA
Watermelon Introduction Watermelon belongs to the genus Citrullus of the subfamily Cucurbitoideae, tribe Benincaseae Ser., Subtribe Benincasinae (Ser.) C. Jeffrey, of the family Cucurbitaceae (Robinson and Decker-Walters 1997). Citrullus was first used as a generic name by Foraskal for this genus in 1775 and then by Schrader (ex Ecklon and Zeyher) during 1834. This genus was included in the list of nomina conservanda by the VIII International Botanical Congress 1954 (Fursa 1972). There are five recognized Citrullus diploid (n = 11) species that naturally grow in xerophytic and semi-arid habitats. Among them, the Citrullus lanatus (Thunb.) Matsum. et Nakai), Citrullus ecirrhosus Cogn., Citrullus rehmii De Winter and Acanthosicycos naudinianus (Sond.) C. Jeffrey are indigenous to the arid sandy regions of southern Africa (Meeuse 1962; Fursa 1972), while C. colocynthis (L.) Schrad. is indigenous to the deserts of North Africa, near East and SouthWest Asia (Bates and Robinson 1995; Robinson and Decker-Walters 1997; Jeffrey 2001). The C. rehmii and C. lanatus are annual species, whereas C. colocynthis (colocynth), C. ecirrhosus and A. naudinianus are perennial species (Jarret and Newman 2000). C. lanatus includes red sweet watermelon group (initially named as C. lanatus subsp. vulgaris and later grouped along with the white flesh types to be broadly classified as C. lanatus var. lanatus) (here onwards will be referred as sweet watermelon because PI 270306 that is in the current study is the red sweet type). According to Jeffrey (2001), the C. lanatus includes both var. lanatus and var. citroides. C. lanatus var. citroides (Bailey) Mansf. ex Greb. (hereafter citron in the current study) is a group of ancient cultigens known as ‘Tsamma’ melons of southern Africa and also called as the ‘cow’ or ‘Citron’ 123 melon (Whitaker and Davis 1962; Whitaker and Bemis 1976; Burkill 1985; Jarret et al. 1997; Laghetti and Hammer 2007; Mujaju et al. 2010). In contrast with the sweet red watermelon varieties, the citron watermelons have white or green flesh with a wide range of flavors, and high pectin and dietary fibers that make them a functional food source for native people in Africa (Dahl Jensen et al. 2011). Among the diverse opinions on the origin and domestication of watermelon, one theory supports that sweet watermelon was derived directly from Citrullus colocynthis and the other theory is about the citron types being the progenitors (Rubatsky 2001). Though PCR RFLP study involving non coding chloroplast DNA revealed that the colocynth is the ancestral progenitor of entire Citrullus genus (Dane and Liu 2007), there are several other marker studies (Jarret et al. 1997; Levi et al. 2001; Nimmakayala et al. 2010) that placed subclusters of citron and sweet melon together paving to the speculation that citron could be the raw material for the domestication of red sweet watermelon. Phylogenies based on the internal transcribed spacer regions of the 18S–28S suggested C. rehmii is closure to the C. lanatus clade as a whole than the colocynth (Jarret and Newman 2000). Sequence phylogenies of Genetic mapping studies using progenies derived from the crosses between citron and sweet watermelon genotypes revealed strong preferential (non-Mendelian) segregation for several markers, indicating that wide structural differences among the types of C. lanatus (Levi et al. 2002, 2006). The current study was designed to resolve relationships among Citrullus spp. using molecular cytogenetic analysis. Fluorescence in situ hybridization (FISH) proved to be useful cytogenetic tool for elucidating genome features and organization, and provided valuable information on homologies between the chromosomal segments of closely related species (Heslop-Harrison 1991). The FISH has been extensively employed in identifying chromosome regions using rDNA sequences, as probes (Leitch and Heslop-Harrison 1992; Adams et al. 2000; Islam-Faridi et al. 2007; Martı́nez et al. 2010). The rDNA markers are known to be one of the robust phylogenetic tools, for inferring genome evolution during speciation (Gu and Xiao 2003; Martı́nez et al. 2010). The current investigation was to study rDNA gains and losses in the speciation of the genus Citrullus. Genet Resour Crop Evol Materials and methods Plant materials and chromosome preparation Seeds of colocynth (PI 386015; collected in Iran), C. rehmii (Grif 16135; collected in southern Africa), sweet watermelon (PI 270306; an Asian type with globular shape watermelon; collected in the Philippines) and citron (PI 244018; collected in South Africa) were obtained from Dr. Robert Jarret at the USDA-ARS, Plant Genetic Resources Conservation Unit, Griffin, GA 30223. These accessions were selected as representative for the different Citrullus spp. since they were used in previous phylogenetic studies using DNA markers (Jarret and Newman 2000; Dane and Liu 2007; Dane et al. 2007; Nimmakayala et al. 2010). Actively growing root tips, about 1 cm long, were excised from young seedlings growing in potting soil in a greenhouse and pre-treated in an aqueous solution of a–monobromonaphthalene (0.8 %, Sigma) for 2 h at room temperature in the dark. The root tips were then fixed in 4:1 (95 % ethanol: glacial acetic acid) to arrest cell division at metaphase. The fixed root tips were washed thoroughly in distilled and 0.01 M citrate buffer prior to processing for enzyme digestion (40 % (v/v) Cellulase (Sigma, USA), 20 % (v/v) Pectinase (Sigma, USA), 3 % (w/v) Cellulase R10 (SERVA, Germany), 2 % (w/v) Cellulase RS (SERVA, Germany), 1 % (w/v) macerozyme (SERVA, Germany), 1.5 % (w/v) Pectolyase Y23 (Kyowa Chemical Products Co., Ltd.) in 0.01 M citrate buffer, and the chromosome spreads were prepared as described elsewhere (Jewell and Islam-Faridi 1994). The chromosome spreads were checked with a phase contrast microscope (AxioImager D1, Carl Zeiss, Inc.) and slides containing good chromosome spreads that are free from cell debris and almost no overlap were selected for FISH and/or stored at -80 C until use for FISH. Germany) using the manufacturer’s instructions. The genomic DNA from citroides was labeled with BioNick-Translation Mix. Fluorescent in situ hybridization (FISH) For FISH, slides previously stored at -80 °C were dehydrated through 70 and 95 % ethanol, 3 min each, and air-dried at least 2 h at room temperature. The slides were then incubated in RNase-A solution (35 lg/ml) in 29 SSC at 37 °C for 1 h, rinsed twice in 29 SSC, 5 min each, and dehydrated in a ethanol series (70, 85 and 95 %, 3 min each) and air-dried overnight prior to FISH. Standard FISH technique was utilized as previously reported (Islam-Faridi et al. 2002). Briefly, the hybridization mixture consisted of 50 % deionized formamide, labeled probe (30 ng/ slide), carrier blocking DNA (E. coli DNA, 7.5 lg/ slide), 10 % dextran sulfate, in 2 9 SSC. Genomic in situ hybridization (GISH) is a molecular cytogenetic technique that employs differential hybridization of entire genomic probes of one species to the chromosome spreads of a counterpart species, facilitating gross assessment of chromosome homology or divergence and physically localizes homologous chromosome regions between related species or subspecies. For GISH, 50 ng of labeled genomic DNA was used for each slide. Total genomic DNA (10–509 of the probe DNA) from cucumber was used as a blocking DNA in the hybridization mixture to prevent cross hybridization. Probe hybridization sites were detected with Cy3-conjugated streptavidin (Jacson Immuno Research Laboratories, West Grove, PA) for biotin labeled probe and FITC-conjugated anti-digoxygenin (Roche, D-68305 Mannheim, Germany). The FISH preparations were counterstained with DAPI (4 lg/ml) in Mcllvaine buffer (pH 7.0) and mounted with Vectashield (Vector Laboratories, CA) to prevent photo-bleaching the fluorochromes when viewing under epi-fluorescence microscope. Probe DNA preparation Digital image capture and process Whole plasmid DNA with 18S–28S rDNA insert of maize (Zimmer et al. 1988) and 5S rDNA insert of sugar beet including the spacer region (Schmidt et al. 1994) were labeled with biotin-16-dUTP (Bio-NickTranslation Mix, Roche, Germany) and/or digoxygenin-11-dUTP (Dig-Nick-Translation Mix, Roche, Digital images were captured using an epi-fluorescence microscope (AxioImager Z1, Carl Zeiss, Germany) with suitable filter sets (chroma Technology, MA, USA) and CoolCube high performance CCD camera, and pre-processed with Ikaros and ISIS V5.1 (MetaSystem Inc., MA, USA). The images were 123 Genet Resour Crop Evol Fig. 1 A bicolor FISH with 18S–28S rDNA (green signals) and 5S rDNA (red signals) probes on watermelon (var. lanatus, PI 270306) chromosome spreads. a Prometaphase chromosome spread. b Late prophase chromosome spread, and c interphase cell then further processed with Adobe Photoshop CS v8 (Adobe System, USA). Results A single root tip yielded several metaphase cells, and slide with at least 20 or more spreads were selected for FISH. The FISH analysis using the 18S–28S rDNA and 5S rDNA probes identified considerable differences in genome organization among the Citrullus species and subspecies in this study (colocynth, sweet watermelon, citron and C. rehmii). In sweet watermelon, two major 18S–28S rDNA sites were found and these were located on two different homologous pairs of chromosomes (Fig. 1a, b, Table 1). A two-color FISH (bicolor FISH) showed that the 5S rDNA site was located interstitially and syntenic to one of the 18S–28S rDNA sites (Fig. 1). The 18S–28S rDNA site and the 5S rDNA site were quite spaced out to each other as Table 1 Number of rDNA (18S–28S and 5S) loci/sites observed in Citrullus spp. Citrullus species 18S–28S rDNA 5S rDNA C. colosynthis 2 (4) 1 (2) C. lanatus, var. lanatus var. citroides C. rehmii F1, var. lanatus 9 var. citroides 2 (4) 1 (2) 1 (2) 2 (4) 1 (2) 1 (2) (3) (3) The total number of FISH signals observed shown in parentheses 123 revealed by the interphase FISH (Fig. 1c). In contrast, only one 18S–28S rDNA site and two 5S rDNA sites were observed in citron. A bicolor FISH analysis in citron showed that 5S rDNA sites were located on two different homologous pairs of chromosomes, while an 18S rDNA site was located on another homologous pair of chromosomes (Fig. 2a, b). The 18S–28S rDNA site that was interstitially located along with one of the 5S rDNA sites on a pair of homologous chromosomes in sweet watermelon appeared to be lost in citron. The FISH revealed the presence of the sweet watermelon chromosome with the 18S–28S rDNA that is colocalized with the 5S rDNA locus, and its counterpart citron chromosome contains only 5S rDNA, but not the 18S–28S rDNA locus (Fig. 3c). A total of six FISH signals, three each of 5S rDNA (Fig. 3a) and 18S–28S rDNA (Fig. 3c) (Table 1) were observed as expected based on the number 5S and 18S sites in sweet watermelon and citron. To elucidate the sweet watermelon versus citron chromosome pairing, probing of 18S–28S rDNA and 5S rDNA loci were performed on root tip spreads of F1 plants [sweet watermelon (PI 270306) 9 citron (PI 244018)]. FISH analysis in F1 spreads revealed the presence of the sweet watermelon chromosome, which had one 18S– 28S rDNA site, co-localized with the 5S rDNA site (Fig. 3c). The citron homologue of this chromosome had only 5S rDNA site indicating deletion of the 18S– 28S rDNA site. The other 18S–28S rDNA site, which is common to both sweet watermelon and citron, was found in homologous pair of F1 chromosomes. The duplicated 5S rDNA site of citron was hemizygous in the F1 spreads (Fig. 3a). Genet Resour Crop Evol Fig. 2 A bicolor FISH with 18S–28S rDNA (green signals) and 5S rDNA (red signals) probes on citroides chromosome spreads. a Mid prophase chromosome spread showing two major 18S– 28S rDNA signals, four 5S rDNA signals, and an interphase nucleus also showing two green signals. b Metaphase chromosome spread showing two major 18S–28S rDNA signals, four 5S rDNA signals Fig. 3 18S–28S rDNA,5S rDNA and GISH sites on F1 (cross of lanatus and citroides). a Single color FISH showing 3 sites of 5S rDNA(green signal), b FISH showing 3 sites of 18S–28S rDNA (green signal) and GISH (red signal). c bicolor FISH with both 5S (green signal) and 18S–28S rDNA sites (red signal) To further substantiate our finding of major chromosomal divergence in citron versus sweet watermelon, we performed GISH on the sweet watermelon (PI 270306) plant chromosomal spreads using the whole genomic DNA of citron (PI 244018) (Fig. 6). The GISH results (Fig. 6) indicated that the citron genome mainly hybridizes to subtelomeric and pericentromeric regions of the sweet watermelon chromosomes, suggesting that these chromosomal regions consist of repetitive DNAs, while greater divergence may exist in genomic regions with low copy gene sequences. The GISH result in the current analysis need to be revalidated as the incomplete hybridization could have caused experimental bias because of improper blocking as the cucumber DNA was used for the blocking. This could be because some of the chromosomal regions in watermelon might have different repetitive elements, which are not shared with the cucumber genome. We performed single and bicolor FISH analysis to understand site number variations of 18S–28S rDNA and 5SrDNA sites in the ancestral species colocynth (PI 386015). The study revealed that the colocynth possessed a similar site number of 18S–28S rDNA and 5SrDNA sites as noted in the sweet watermelon, indicating that the cultivated watermelon has similar chromosomal organization to the ancestral progenitor colocynth (Fig. 4). 123 Genet Resour Crop Evol Fig. 4 FISH probed with 18S–28S and 5S rDNA clones on C. colocynthis metaphase chromosome spread. a Species colocynthis contained two sites of 18S–28S rDNA (red signals) and they are on two different homologous pairs of chromosomes. b FISH showing a site of 5S rDNA (green signals) and located interstitially and syntenic to one of the 18S–28S rDNA (may be the robust one) site. c superimposed image of ‘‘a’’, ‘‘b’’ and ‘‘d’’. d chromosome spread of C. colocynthis stained with DAPI Fig. 5 FISH probed with 18S–28S and 5S rDNA clones on rehmii chromosome spread. a Species rehmii contained one site each of 18S–28S rDNA (red signals) and 5S rDNA (green signals) located on two different chromosomes FISH analysis was carried out on the root tip chromosome spreads of C. rehmii to note that this species contained only one each of 18S–28S rDNA and 5S rDNA sites (Fig. 5). Apparently, as noticed in FISH analysis of citron, the 18S–28S rDNA site that was observed to be syntenic to the 5S rDNA site as in colocynth and sweet watermelon also lost in C. rehmii. 123 The duplication event of the 5S rDNA site that was observed in var. citroides was not found in C. rehmii, indicating that the duplication of the 5S rDNA site in citron could be relatively recent (Fig. 6). Results pertaining to major structural rearrangements and site number variation in various species of the genus Citrullus are summarized in a flow chart (Fig. 7). Genet Resour Crop Evol Fig. 6 Genomic DNA in situ hybridization using citroides DNA as probe on F1 Fig. 7 Flow chart showing rDNA distribution between C. colocynthis, C. lanatus var. lanatus, C. rehmii and C. lanatus var. citroides Discussion Physical mapping and characterization of 18S–28S rDNA and 5S rDNA sites have provided a number of useful chromosome markers in several plant species (Hizume et al. 1992; Brown and Carlson 1997; IslamFaridi et al. 2007). PCR selection and variation in site number among polymorphic sequences present in a genome can lead to a biased representation for different paralogues (Álvarez and Wendel 2003; Fukushima et al. 2008). Current FISH analyses in Citrullus accessions were undertaken to eliminate PCR bias and characterize site number of 18S–28S rDNA and 5S rDNA. In the current study, the FISH analysis demonstrated that the rDNA gene loci (18S–28S and 5S) are useful cytogenetic markers, showing wide differences among the Citrullus species and subspecies. Dane and Lang (2004) and Jarret and Newman (2000) proposed that C. rehmii might be the ancestral to sweet watermelon. The results in this study clearly showed the close relationship of C. rehmii with both citron and sweet watermelon complementing previously assumed phylogenetic relationship. Similar to the other diploid plant species (Hanson et al. 1996; Linares et al. 1996; Murata et al. 1997), our research revealed that the number of major 18S–28S rDNA loci in Citrullus genus ranged between 1–4, and the 5S rDNA loci tended to be localized at one or two positions. This study confirmed that the FISH is a powerful tool that can be useful to further study the Citrullus spp. chromosomes, possibly by using low copy BACs or other protein coding gene families, as has been shown in other plant species, such as Iris (Martı́nez et al. 2010). In sweet watermelon and colocynth, our study has shown that the two major 18S–28S rDNA loci are on different chromosomes, while the 5S rDNA locus positioned interstitially and syntenic to one of the 18S–28S rDNA loci (Hanson et al. 1996). The two major 18S–28S rDNA sites of sweet watermelon and colocynth, which were found on two 123 Genet Resour Crop Evol different homologous pairs, are not located in citron in the same configuration. On the contrary, only one 18S–28S rDNA site was found in citron and C. rehmii. The 18S–28S rDNA site that was interstitially located along with the 5S rDNA site in sweet watermelon and colocynth was apparently deleted in citron and C. rehmii. The biological significance of this loss and its potential adaptive significance is unclear. FISH analysis of sweet watermelon and citron F1 progeny revealed occurrence of uniparental homologous rDNA gene copies pertaining to sweet watermelon chromosome with the 18S–28S rDNA site, which is closely linked to a 5S rDNA site and the citron homoeologue of this chromosome that had only 5S rDNA site showed the deletion of the 18S–28S rDNA site. Many earlier studies indicated that the 18S–28S sites present differentially in closely related species, which may be because of changing the positions (Berjano et al. 2009). In our study, we identified polymorphic levels of 5S rDNA sites because of the apparent duplication of these sites in citron. Previous studies indicated the 5S rDNA site is a stable chromosome marker (PedrosaHarand et al. 2006; Berjano et al. 2009). The duplication event of 5S rDNA site in citron should be further analyzed using PCR studies to characterize the intra and interspecific length variation as well as to categorize pseudo and functional 5S gene units. The 5S rDNA is generally crossing over causing molecular drives for genome evolution (Ohta 1984; Dover 1986). Classical taxonomic study (Robinson and DeckerWalters 1997), molecular markers (Jarret et al. 1997; Levi et al. 2001; Nimmakayala et al. 2010); isozymes (Zamir et al. 1984; Navot and Zamir 1987), internal transcribed spacer regions (ITS1 and ITS2) of 18S– 28S nuclear ribosomal DNA (Jarret and Newman 2000) and detailed chloroplast analysis (Dane and Lang 2004; Dane et al. 2004; Dane and Liu 2007) established that the cultivated watermelon is phylogenetically sister to citron. All the species in Citrullus genus are freely crossable in varying degrees, producing semi sterile to fertile progenies, which made elucidation of domestication process in cultivated watermelon complicated to understand. Our cytogenetic analysis showed that sweet watermelon has exactly the same rDNA distribution, number of loci and configuration as in the ancestral species colocynth, and differs drastically when compared with its sister 123 citron. This result highlights the chromosome structural differences in rDNA site number variation between citron and sweet watermelon, explaining why preferential segregation could occur in F2 and BC1 populations derived from crosses between sweet watermelon 9 citron, indicating that broad differences may exist in genome organization of these two subspecies (Levi et al. 2002, 2006). The accessions of various taxa used in the current research were chosen based on their use by previous researchers for analysis of chloroplast phylogenies and molecular markers. The citron accession PI 244018 with cream color seeds with black and grayish black spots from Transvaal, South Africa was characterized as chloro type 3 by Dane and Liu (2007). The cultivated sweet watermelon accession PI 270306 (Philippines) with red flesh color and black seeds did not cluster with citron and colocynth, when subjected to principal component analyses using a large number of shared markers, indicating it could be a representative model of the sweet watermelon taxon (Nimmakayala et al. 2010). Colocynth (PI 386015) from Iran was characterized to possess haplotype II of G3pdh (Dane et al. 2007), and the C. rehmii (Grif 16135) accession was the same as used by Jarret and Newman (2000) for internal transcribed spacer sequence analysis to conclude C. rehmii was the progenitor for C. lanatus. In particular, the number and localization of 5S rDNA loci is often a distinguishing feature between even closely related genomes, as has been shown by ecotype specific polymorphisms in Arabidopsis thaliana (Fransz et al. 1998). Based on this, we conclude the present study should be extended to the other characterized chlorotypes (citron 1 and citron 2) of citron (Dane and Liu 2007) as well as colocynth accessions from various phylogeographic clusters (Dane et al. 2007) and sweet watermelon accessions that were characterized to belong to different clusters (Nimmakayala et al. 2010). The information pertaining to rDNA site distribution and number variation will help to choose parents from various species for introgression of wild genes into cultivated watermelon. Acknowledgments The authors are grateful to Dr. Jarret, Plant Genetic Resources Conservation Unit, USDA-ARS, Griffin, GA, 30223 for providing the seeds of germplasm accessions. 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