INVESTIGATION Evidence for Emergence of Sex-Determining Gene(s) in a Centromeric Region in Vasconcellea parviflora Marina Iovene,*,† Qingyi Yu,‡ Ray Ming,§,** and Jiming Jiang*,1 *Department of Horticulture, University of Wisconsin, Madison, Wisconsin 53706, †Consiglio Nazionale delle Ricerche–Institute of Biosciences and BioResouces, Bari 70126, Italy, ‡Department of Plant Pathology and Microbiology, Texas A&M AgriLife Research Center, Texas A&M University System, Dallas, Texas 75252, §Department of Plant Biology, University of Illinois at Urbana–Champaign, Urbana, Illinois 61801, and **Fujian Agriculture and Forestry University, Fuzhou, Fujian, 350002, China ABSTRACT Sex chromosomes have been studied in many plant and animal species. However, few species are suitable as models to study the evolutionary histories of sex chromosomes. We previously demonstrated that papaya (Carica papaya)(2n =2x = 18), a fruit tree in the family Caricaceae, contains recently emerged but cytologically heteromorphic X/Y chromosomes. We have been intrigued by the possible presence and evolution of sex chromosomes in other dioecious Caricaceae species. We selected a set of 22 bacterial artificial chromosome (BAC) clones that are distributed along the papaya X/Y chromosomes. These BACs were mapped to the meiotic pachytene chromosomes of Vasconcellea parviflora (2n =2x = 18), a species that diverged from papaya 27 million years ago. We demonstrate that V. parviflora contains a pair of heteromorphic X/Y chromosomes that are homologous to the papaya X/Y chromosomes. The comparative mapping results revealed that the male-specific regions of the Y chromosomes (MSYs) probably initiated near the centromere of the Y chromosomes in both species. The two MSYs, however, shared only a small chromosomal domain near the centromere in otherwise rearranged chromosomes. The V. parviflora MSY expanded toward the short arm of the chromosome, whereas the papaya MSY expanded in the opposite direction. Most BACs mapped to papaya MSY were not located in V. parviflora MSY, revealing different DNA compositions in the two MSYs. These results suggest that mutation of gene(s) in the centromeric region may have triggered sex chromosome evolution in these plant species. KEYWORDS FISH; centromere; heterochromatin; sex chromosome ETEROMORPHIC sex chromosomes (X/Y, Z/W) evolved homologous XY systems and all birds have homologous ZW Hfrom pairs of autosomes (Ming et al. 2011; Bachtrog systems (Graves 2008). Thus, these sex chromosomes, sur- 2013; Charlesworth 2013). It is generally accepted that sex prisingly, appear to have evolved only once in each of these chromosome evolution is initiated by the emergence of a sex- two major evolutionary clades. determining locus. Suppression of recombination surround- Compared to mammals and birds, sex chromosomes have ing the sex-determining locus, which favored the linkage of emerged more frequently in several other eukaryotic lineages, the sex-determining alleles with sexually antagonistic alleles, including plants. Sex chromosomes have been reported in at caused the decay of the Y/W chromosomes and resulted in least 48 plant species across 20 different families, including a pair of heteromorphic sex chromosomes (Ming et al. 2011; both X/Y and Z/W systems (Ming et al. 2011; Kumar et al. Bachtrog 2013; Charlesworth 2013). Sex chromosomes evolved 2014). Sex chromosomes in some plant species were identi- independently in different eukaryotic lineages. The mammalian fied using genetic mapping-based approaches. However, cyto- X chromosome and the bird Z chromosome evolved from dif- genetic data are not available from most of these species. Thus, ferent portions of the ancestral genome (Bellott et al. 2010; it is unclear whether heteromorphic chromosomes have al- Cortez et al. 2014). Interestingly, all therian mammals have ready developed in these species, such as asparagus (Asparagus officinalis) (Telgmann-Rauber et al. 2007), Populus (Populus Copyright © 2015 by the Genetics Society of America trichocarpa)(Yinet al. 2008), and spinach (Spinacia oleracea) doi: 10.1534/genetics.114.173021 (Yamamoto et al. 2014). Heteromorphic sex chromosomes Manuscript received October 4, 2014; accepted for publication November 26, 2014; published Early Online December 5, 2014. were revealed cytologically in several plant species (Yamato Supporting information is available online at http://www.genetics.org/lookup/suppl/ et al. 2007; Zhang et al. 2008; Sousa et al. 2013), including, doi:10.1534/genetics.114.173021/-/DC1. 1Corresponding author: Department of Horticulture, 1575 Linden Dr., Room 409A, Silene latifolia, which is one of the best-studied model plant University of Wisconsin, Madison, WI 53706. E-mail: [email protected] species with well-differentiated X/Y chromosomes (Vyskot Genetics, Vol. 199, 413–421 February 2015 413 and Hobza 2004; Macas et al. 2011). Multiple sex chromo- somes, such as XY1Y2, were also reported in several plant species (Hizume et al. 1988; Howell et al. 2009; Mariotti et al. 2009; Navajas-Perez et al. 2009). Most of the plant species with sex chromosomes have not been sequenced. We have limited knowledge of the evolutionary histories of plant sex chromosomes because of scarce genomic resources and a lack of cytological and comparative studies of geneti- cally related plant species with sex chromosomes. Most species in the Caricaceae family are dioecious. Therefore, dioecy likely represents the ancestral sexual state in this family (Charlesworth 2013). Papaya (Carica papaya) (2n =2x = 18), an important fruit crop in Caricaceae, con- tains a pair of X/Y chromosomes that emerged only a few million years ago (MYA) (Yu et al. 2008; Wang et al. 2012). The young nature of papaya sex chromosomes was confirmed cytologically in which only a small portion (13%) of the X/Y Figure 1 Phylogenetic relationships in family Caricaceae. Branch lengths are proportional to time. Numbers on nodes represent age in million chromosomes is cytologically differentiated (Zhang et al. 2008). years. This chronogram is adapted from Carvalho and Renner (2012). Thus, papaya provides a model system to capture early molec- Species sampled in this study are in boldface type. ular and cytological events during sex chromosome evolution. The Vasconcellea/Jacaratia clade in Caricaceae is closely related or digoxigenin-11-dUTP (Roche Diagnostics, Indianapolis, IN), to papaya (Carvalho and Renner 2012) (Figure 1). We were using a standard nick translation reaction. Chromosomes were intrigued to know whether the dioecious Vasconcellea/Jacaratia counterstained with 49,6-diamidino-2-phenylindole (DAPI) in species contains sex chromosomes and whether such sex chro- VectaShield antifade solution (Vector Laboratories, Burlingame, mosomes are related to the papaya X/Y chromosome. We con- CA).TheFISHimageswereprocessedwithMetaImagingSe- ducted fluorescence in situ hybridization (FISH) of a set of ries 7.5 software. The final contrast of the images was processed bacterial artificial chromosome (BAC) clones, all of which using Adobe Photoshop CS3 software. Heterochromatin length were previously mapped to the papaya X/Y chromosomes, in was estimated based on DAPI staining pattern of the putative Y two Caricaceae species, Vasconcellea parviflora (2n =2x = 18) chromosome and expressed in percentage of the total chromo- and Jacaratia spinosa (2n =2x = 18). We demonstrate that some length. The Y-specific heterochromatic domain was esti- V. parviflora contains a pair of heteromorphic X/Y chromo- mated as the difference in heterochromatin content between somes that are homologous to the papaya X/Y chromosomes. X and Y chromosomes and was expressed as the percentage By contrast, the same pair of chromosomes in J. spinosa is of the total chromosome length. The position of a BAC clone homomorphic. These results reveal a dynamic nature of sex or other landmarks along the chromosome was estimated and chromosome evolution in the Caricaceae species. expressed as D/L 3 100, where D = distance of the landmark from the end of the short arm, and L = total chromosome Materials and Methods length. All measurements were made on digital photographs, using Meta Imaging Series 7.5 software. Materials fl Plants of V. parvi ora and J. spinosa were maintained at the Results Hawaii Agriculture Research Center as well as in the Texas fi fl AgriLife Research Center in Weslaco, Texas. Young male Identi cation of the X/Y chromosomes in V. parvi ora fl fi ower buds were collected and xed in 3:1 (100% ethanol: V. parviflora is a dioecious species with female and male ’ 2 ° glacial acetic acid) Carnoy s solution and kept at 20 until individuals. Both male and female flowers are morphologi- use. Twenty-nine papaya BAC clones were previously selected cally similar to those of papaya (Figure 2). Young flower fi and were either speci c to the papaya sex-determining region buds were collected from male V. parviflora plants to prepare fi (X or Y speci c) or to the papaya sex chromosomes (Zhang meiotic pachytene chromosomes. The pachytene karyotype of et al. 2008; Wai et al. 2012). A telomeric DNA probe, pAtT4 V. parviflora generally resembled the papaya karyotype (Zhang from Arabidopsis thaliana (Richards and Ausubel 1988), was et al. 2010), containing nine metacentric and submetacentric fl used to label the ends of V. parvi ora pachytene chromosomes. chromosomes of similar sizes (Figure 3C). Heterochromatin, which is brightly stained by DAPI, was visible in the pericentro- FISH meric regions of all nine pachytene chromosomes (Figure 3C). Chromosome