Chromosome Res DOI 10.1007/s10577-017-9555-y ORIGINAL ARTICLE Cytogenetic comparison of heteromorphic and homomorphic sex chromosomes in Coccinia (Cucurbitaceae) points to sex chromosome turnover Aretuza Sousa & Jörg Fuchs & Susanne S. Renner Received: 11 January 2017 /Revised: 17 February 2017 /Accepted: 27 February 2017 # Springer Science+Business Media Dordrecht 2017 Abstract Our understanding of the evolution of plant in male cells of all three species accumulate some of the sex chromosomes is increasing rapidly due to high- very same repeats that are enriched on the C. grandis Y throughput sequencing data and phylogenetic and chromosome, pointing to either old (previous) sex chro- molecular-cytogenetic approaches that make it possible mosomes or incipient (newly arising) ones, that is, to sex to infer the evolutionary direction and steps leading from chromosome turnover. A 144-bp centromeric satellite homomorphic to heteromorphic sex chromosomes. Here, repeat (CgCent) that characterizes all C. grandis chro- we focus on four species of Coccinia,agenusof25 mosomes except the Y is highly abundant in all centro- dioecious species, including Coccinia grandis,thespe- meric regions of the other species, indicating that the cies with the largest known plant Y chromosome. Based centromeric sequence of the Y chromosome diverged on a phylogeny for the genus, we selected three species very recently. close to C. grandis to test the distribution of eight repet- itive elements including two satellites, and several plastid Keywords Coccinia species . genome size . FISH . and mitochondrial probes, that we had previously found histone modification . repetitive DNA . plant sex to have distinct accumulation patterns in the C. grandis chromosomes genome. Additionally, we determined C-values and per- formed immunostaining experiments with (peri-)cen Abbreviations tromere-specific antibodies on two species (for compar- BAC Bacterial artificial chromosome ison with C. grandis). In spite of no microscopic chro- FISH Fluorescent in situ hybridization mosomal heteromorphism, single pairs of chromosomes PCR Polymerase chain reaction rDNA Ribosomal DNA Responsible Editor: Hans de Jong. SDR Sex-determining region Electronic supplementary material The online version of this article (doi:10.1007/s10577-017-9555-y) contains supplementary material, which is available to authorized users. Introduction A. Sousa (*) : S. S. Renner (*) Department of Biology, University of Munich (LMU), 80638 Munich, Germany In flowering plants, morphologically distinct e-mail: [email protected] (heteromorphic) sex chromosomes are known from 19 e-mail: [email protected] species in 4 families, while homomorphic sex chromo- J. Fuchs somes are known from at least 20 species in 13 families, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), including both X/Yand Z/W systems (Ming et al. 2011; Gatersleben, 06466 Stadt Seeland, Germany Charlesworth 2016). Their patchy phylogenetic Sousa A. et al. distribution implies that most angiosperm sex chromo- chromosomes of the distant papaya relative some systems evolved independent of each other. The Vasconcellea parviflora, while some of the same BACs few systems for which densely sampled phylogenies are applied in Jacaratia spinosa, another distant relative of available further have shown that even species in the papaya, were located at regions showing perfect pairing same genus, such as Silene colpophylla and Silene otitis, at the pachytene stage without any loop or sign of which last shared a common ancestor 10–11 million heteromorphism. years ago (Slancarova et al. 2013), have non- Here, we analyze whether any Coccinia grandis Y homologous sex chromosomes, the first in an XY sys- chromosome-enriched sequences can be used to identify tem and the second in a ZW system (Mrackova et al. (potential) homomorphic sex chromosomes in its close 2008;Maraisetal.2011; Slancarova et al. 2013). Spe- relatives. In C. grandis, the accumulation of repetitive cies with homomorphic sex chromosomes presumably elements (as well as plastid and mitochondrial DNA) on have small sex-determining regions (SDRs), as inferred the Y chromosome causes a male/female difference of for Vitis vinifera (Fechter et al. 2012;Picqetal.2014), 100 Mbp/2C (Sousa et al. 2013), which raises the ques- Populus trichocarpa and P. tremuloides (Geraldes et al. tion if the same elements are already abundant in related 2015), Actinidia chinensis (Zhang et al. 2015), Carica species (Piednoel et al. 2012). To date, there are no papaya (Wang et al. 2012; Lappin et al. 2015), and experimental or other studies on the sex determination Fragaria chiloensis (Tennessen et al. 2016). The sex system in any of the species of Coccinia other than chromosomes in these species may be between 18 and 2 C. grandis. The latter species is native to tropical Africa million years old (Wang et al. 2012;Picqetal.2014; and India and a member of a small, entirely dioecious Geraldes et al. 2015;Lappinetal.2015; Tennessen et al. Cucurbitaceae genus of 25 species (Holstein and Renner 2016), but most these estimates assume that the respec- 2011;Holstein2015). So far, C. grandis is the only tive sex chromosomes are as old as the genus or subge- species in the genus known to have heteromorphic sex nus to which the focal species belongs, which may be a chromosomes. The Y chromosome is so large that the risky assumption. The small SDRs of homomorphic sex male C. grandis genome is 10% larger than the female chromosomes may result from occasional XY recombi- (Sousa et al. 2013). The sex chromosomes of C. grandis nation and from a high rate of turnover, meaning that sex are about 3 Ma old (Holstein and Renner 2011) and are chromosomes are replaced before they had time to de- unusual among flowering plant sex chromosomes in the cay (Tennessen et al. 2016). For example, the SDR of Y being heterochromatic (Sousa et al. 2013, 2016). P. trichocarpa sits on the subtelomeric region of chro- Using a phylogeny that includes most of the 25 species mosome 19 (Geraldes et al. 2015)andthatof of Coccinia,weselectedC. hirtella, C. sessilifolia,and P.tremuloides on the pericentromeric region of the same C. trilobata as closely related to C. grandis and then chromosome (Kersten et al. 2014), which suggests applied molecular cytogenetic methods, as well as C- restructuring of the sex chromosomes in these closely value comparisons of male and female genomes. The related species. questions that we wanted to answer were as fol- Most studies of homomorphic sex chromosomes lows: (i) what is the extent of variation in chro- have focused on the construction of genetic maps in mosome numbers and/or genome sizes among silico for the identification of X-Yor Z-W-linked homo- close relatives of C. grandis? (ii) Are potential logues (Asparagus officinalis: Telgmann-Rauber et al. chromosome number deviations accompanied by 2007; Deng et al. 2012; Harkess et al. 2015; Silene variations in the number of ribosomal DNA otites: Slancarova et al. 2013; A. chinensis:Fraser (rDNA) loci or the presence of interstitial et al. 2009 ; Fragaria virginiana: Spigler et al. 2008; telomeric sequences? (iii) Do any chromosome Tennessen et al. 2016; Populus hybrids: Yin et al. 2008; pairs differentially accumulate repeats (a question Pistacia vera: Kafkas et al. 2015). Such markers have for which we focused on those repeats accumulat- rarely been tested in situ, except in homomorphic sex ed on the heteromorphic sex chromosomes of chromosomes of C. papaya (Zhang et al. 2008). Taking C. grandis) or have an extended centromere- advantage of 29 bacterial artificial chromosomes specific histone marker accumulation, as found in (BACs) developed to cover the papaya sex- the C. grandis Y chromosome? And (iv) is the determining region, Iovene et al. (2015) detected a 144-bp-long centromere satellite that in C. grandis minute heteromorphic region in pachytene meiotic hybridizes to the centromeres of all chromosomes Heteromorphic and homomorphic sex chromosomes except the Y also detectable in other species of generate CocA1, and its purified PCR product was Coccinia? Rapid divergence of sex chromosome labeled with digoxigenin-11-dUTP (Roche) by nick centromeres is known from primates in which the translation. Fluorescence in situ hybridization, image X chromosomes of closely related species can capture, and editing were performed as Sousa et al. differ in just their centromeric position (Ventura (2016). et al. 2001). Chromosome preparation for FISH Materials and methods Root tips of C. hirtella and C. sessilifolia were collected from potted plants and pretreated in 70 ppm of cyclo- Plant material heximide (Roth) in 2 mM 8-hydroxyquinoline for 5 h at 18 °C to obtain higher numbers of metaphase cells Tubers of C. hirtella, C. sessilifolia,and (Tlaskal 1980). Root tips were fixed in freshly prepared C. trilobata, all of them dioecious climbers, were 3:1 (v/v) ethanol/glacial acetic acid at room temperature collected by N. Holstein in Northeastern Tanzania, for 2 h, transferred to 70% ethanol, kept at room tem- Africa, in 2009. In Munich, we are cultivating these perature overnight, and afterwards stored at −20 °C until plants in the greenhouses of the Botanical Garden, use. Cell suspensions for dropping were obtained fol- and vouchers have been deposited in the herbarium lowing the protocol of Aliyeva-Schnorr et al. (2015) of Munich (official acronym M; for voucher list, see with minor modifications as in the study by Sousa Holstein (2015;hisTable2)). Pistillate or staminate et al. (2016). To make meiotic slides of C. trilobata, flowers were used to identify female or male plants. we used the squashing technique as described in Sousa et al. (2013). FISH probes and conditions Immunofluorescence Arabidopsis-type telomeres and 5S and 45S rDNA sites were visualized according to Sousa et al. (2013), with Immunostaining experiments were carried out on the addition of an ethanol series of 70-90-100%, 2 min C. hirtella and C. sessilifolia. Root tips were pretreated each, after the final washes.