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Home , Jacaratia, Vasconcellea parviflora, Y chromosome

INVESTIGATION

Evidence for Emergence of -Determining (s) in a Centromeric Region in 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 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 have been studied in many plant and animal . However, few species are suitable as models to study the evolutionary histories of sex chromosomes. We previously demonstrated that ( papaya)(2n =2x = 18), a fruit tree in the family , 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 (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 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 of gene(s) in the centromeric region may have triggered evolution in these plant species.

KEYWORDS FISH; centromere; ; sex chromosome

ETEROMORPHIC sex chromosomes (X/Y, Z/W) evolved homologous XY systems and all birds have homologous ZW Hfrom pairs of (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 . Suppression of recombination surround- Compared to 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 with sexually antagonistic alleles, including . 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- and the bird Z chromosome evolved from dif- genetic data are not available from most of these species. Thus, ferent portions of the ancestral (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 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/ 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 (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 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 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 preparation and FISH followed previously Surprisingly, a large loop was consistently observed on one of published protocols (Cheng et al. 2002; Iovene et al. the pachytene chromosomes (Figure 3A). The two homologous 2008). BAC DNA was labeled with either biotin-16-dUTP chromosomes were separated and unpaired within the loop.

414 M. Iovene et al. Figure 2 Morphology of flowers from papaya and V. parviflora. (A) Papaya male flowers. (B) Papaya female flowers, which occur individually or in clus- ters. (C) V. parviflora male flowers. (D) V. parviflora female inflorescence, which usually contains .10 flowers.

A large and a small heterochromatic domain were observed on contains five knobs, including one large knob shared by X only one of the two homologs in the loop (Figure 3B). and Y and four small knobs specific to the papaya MSY (Zhang We next conducted FISH analysis on V. parviflora pachy- et al. 2008). The loop associated with the V. parviflora X/Y tene cells, using several BAC clones that were previously chromosomes is located at a similar position to that of the MSY mapped to the papaya X/Y chromosomes. Most of these onthepapayaYchromosome(Figure5).Thelengthofthe BACs were mapped to this loop-associated V. parviflora loop accounted for 12% (11.7 6 3.04, n =15)ofthe chromosome pair, suggesting that they are homologous to V. parviflora Y chromosome. In comparison, the MSY of pa- the papaya X/Y chromosomes (Figure 3C). Therefore, this paya accounts for 13% of the papaya Y chromosome (Zhang pachytene chromosome pair is likely the sex chromosome et al. 2008). However, more heterochromatin has accumu- pair in V. parviflora and the loop region represents the lated in the V. parviflora MSY than in the papaya MSY. No MSY. Significant accumulation of heterochromatin and pos- loop was observed in the papaya MSY, although the Y chro- sibly chromosomal rearrangements, such as inversions and/ mosome, which contains 8.1 Mb DNA compared to 3.5 Mb for or duplications, within the loop may prevent pairing of the the corresponding X region (Wang et al. 2012), has to twist putative X and Y chromosomes in this region. (zigzag) in pairing with the X within the MSY (Zhang et al. 2008). In comparison, the V. parviflora Y chromosome Distinct heterochromatin distribution patterns associated with the V. parviflora X/Y chromosomes appeared to have accumulated much more DNA than the X chromosome in the loop. Self “pairing” or “supercoiling” of We prepared pachytene chromosomes at both early and late the Y chromosome was observed within the loop on early stages to further investigate differentiation between pachytene chromosomes (Figure 4A). the X and Y chromosomes. Two large heterochromatin blocks The centromere of the papaya Y chromosome was mapped (B), B1 and B2, and three small heterochromatic knobs (K), close to knob 4 within the MSY (Zhang et al. 2008) (Figure K1, K2, and K3, were observed on the X/Y chromosomes 5). The chromosomal domain between B1 and K1 in the (Figure 4). These heterochromatic domains were best visual- V. parviflora MSY was consistently less stained and appeared ized on middle pachytene chromosomes (Figure 4B). B1 and morphologically to be the primary constriction. Based on this K1 were observed only on one of the two homologs, most structure, as well as the synteny between papaya X/Y and likely the Y chromosome. B1 was consistently located in the V. parviflora X/Y chromosomes (see below), the centromere loop on both early and late pachytene chromosomes. K1 was of the V. parviflora Y chromosome is predicted to be located much smaller than B1, but was detectable within the loop on between B1 and K1 within the loop (Figure 5). most pachytene chromosomes. Heterochromatic domains Comparative FISH mapping of the X/Y chromosomes in were often divided into two or more separate subdomains papaya and V. parviflora on highly extended early pachytene chromosomes (Figure 4A). The heterochromatin distribution pattern on V. parviflora We selected a total of 29 BAC clones that were previously X/Y chromosomes is similar to the pattern observed on papaya mapped to the papaya X/Y chromosomes (Zhang et al. 2008; X/Y chromosomes. The papaya X/Y pachytene chromosome Wai et al. 2010; Na et al. 2012), including 11 BACs specific

Sex Chromosomes in V. parviflora 415 Figure 3 Pachytene chromosomes of V. parviflora. (A) A complete pachytene of V. parviflora. The green FISH signals were derived from an Arabidopsis telo- meric DNA probe pAtT4. The red FISH signal (arrow) was derived from BAC 1, which is specific to papaya X/ Y chromosomes. The loop on the X/Y chromosome pair is included in a white box. Bar, 10 mm. (B) The DAPI-stained chromosomes in A were converted into a black-and-white image to enhance the visualization of heterochromatin. The X (red) and Y (green) chro- mosomes within the loop are illustrated in the inset. A large Y-specific heterochromatin domain in the loop is illustrated in blue. (C) A pachytene karyotype devel- oped from the same cell as in A. Individual chromosomes were digitally isolated, straightened, and arranged based on their length, except that the X/Y chromosome pair wasarrangedasthefirst. The arrow points to the FISH signal derived from BAC 1.

to X, 1 BAC specific to Y, and 17 BACs shared by X and Y almost the entire long arm, occurred in one of the two (Supporting Information, Table S1). These BACs were map- species after divergence from a common ancestor. The re- ped to the homologous V. parviflora chromosomes by FISH verse order of BACs 13 and 14 in the two species (Figure 5) to further examine the syntenic relationship of the sex chro- also supports this inversion hypothesis. Interestingly, BACs mosomes in the two species. Only 22 papaya BACs gener- 15–21 maintained the same order on the long arm in the ated distinct FISH signals on V. parviflora chromosomes. two species (Figure 5 and Figure 6E). Thus, an indepen- These clones were labeled in numbers from 1 to 22 (Figure 5, dent and smaller inversion (inversion 2 in Figure 5) spanning Table S1). BACs 15–21 occurred within the chromosomal domain Five BACs, including clones 8–11 that are located in the spanned by inversion 1. papaya MSY, were surprisingly mapped to the distal region BACs 1–6, which span the short arm of papaya X/Y chro- on the long arm of V. parviflora X/Y chromosomes (Figure 5). mosomes, maintained the same order on V. parviflora X/Y By contrast, BAC 22, the most distal clone on the long arm of chromosomes (Figure 5 and Figure 6A). However, the dis- papaya X/Y chromosomes, was mapped to the middle of the tance between BAC 1 and the was much shorter in V. parviflora X/Y chromosomes (Figure 5 and Figure 6C). papaya than in V. parviflora, suggesting another inversion in Thus, an inversion (inversion 1 in Figure 5), which spans this region (inversion 3 in Figure 5).

Figure 4 Heterochromatin distribution along the V. parviflora X/Y chromosomes. (A) An early pachytene chromosome. Two separated subdo- mains are visible from K1 and K2. The B1 region is self-supercoiled. (B) A middle pachytene chro- mosome. All five heterochromatin domains are clearly visible. B1 and K1 are located in the loop and are associated with only one of the two homologs. (C) A late pachytene chromosome. The chromosomes were stained by DAPI and were converted into black-and-white images. The five heterochromatic blocks (B1, B2) or knobs (K1, K2, K3) are indicated by red arrow- heads and arrows. Bars, 10 mm.

416 M. Iovene et al. (Figure S1). By contrast, massive amounts of 5S rDNA-related sequences were amplified in the papaya MSY (Zhang et al. 2010). Based on the position of the Y chromosome centromere within the MSY, the majority of the papaya MSY is located on the long arm of the Y chromosome (Zhang et al. 2008). In contrast, the heterochromatic domain B1 represents more than half of the V. parviflora MSY (Figure 4). This region, spanned by BACs 5 and 6, was significantly expanded in V. parviflora compared to the same region in papaya (Figure 5). Since the centromere of the V. parviflora Y chromosome is located between K1 and B1, the majority of the V. parviflora MSY is located on the short arm of the Y chromosome (Figure 5). Thus, the papaya MSY has expanded toward the long arm of the Y chromosome, whereas the V. parviflora MSY has expanded toward the short arm of the Y chromosome. J. spinosa chromosomes homologous to papaya X/Y chromosomes are homomorphic Jacaratia is one of six genera in Caricaceae. Phylogenetic analysis revealed that Jacaratia and Vasconcellea species are equally distant from papaya (Carvalho and Renner 2012) (Figure 1). We were interested whether Jacaratia species also shared the same sex chromosomes with papaya and V. parviflora. We chose one dioecious species, J. spinosa (2n =2x = 18), for comparative FISH mapping. We conducted FISH analysis, using nine papaya BAC clones (BACs 3 and 5–12) (Table S2)onJ. spinosa chro- mosomes. The chromosomal positions of all nine BACs in J. spinosa were similar to those observed on V. parviflora Figure 5 Comparative mapping of the X/Y chromosomes in papaya X/Y chromosomes, including the distal locations of BACs (adapted from Zhang et al. 2010; Wai et al. 2012) and V. parviflora. BACs – in red numbers in boldface type are specific to the papaya X chromo- 8 12 (Figure 7 and Table S2). Therefore, the three inversions some. BAC 11* generated signals on an additional V. parviflora chromo- associated with this chromosome in Caricaceae species are some. BAC 7** was mapped to a different chromosome in V. parviflora. not specifictoV. parviflora and may have emerged during The green dashed lines mark the putative positions of the on the evolution of the papaya genome. the Y chromosomes. The position of the papaya MSY and the loop on Surprisingly, the J. spinosa chromosome pair that is ho- V. parviflora Y chromosomes are marked. Three inversions are predicted fl based on the order and position of BAC clones in the two species. The mologous to the papaya/V. parvi ora X/Y chromosomes different locations of BAC 1 in the two species are illustrated by a black showed perfect pairing at the pachytene stage without any dashed line. Blue and red dashed lines highlight the centromeric vs. loop. No chromatin differentiation between the two homo- telomeric positions of the four BACs in the two species. Note that the logs was observed on this pachytene chromosome (Figure 7, three inversions drawn along the V. parviflora Y chromosome likely B and D). We did not observe any heteromorphic pachytene emerged during the evolution of papaya. chromosome in J. spinosa. However, it is not conclusive re- garding the absence of heteromorphic sex chromosomes in Comparative FISH mapping of the MSYs in papaya and this species because we were able to collect and analyze only V. parviflora few meiotic cells at the pachytene stage. Although the two MSYs shared a similar chromosomal location and size, they differed in structure and DNA composition. The Discussion chromosomaldomainspannedbyBACs8–11 represented a sig- nificant part of the papaya MSY. This region, however, was Sex chromosomes emerged multiple times independently in located at the distal end of the long arm of V. parviflora X/Y several eukaryotic lineages, including fishes, , and chromosomes (Figure 5). In addition, BAC 7 was not mapped plants (Graves 2008; Ming et al. 2011; Bachtrog 2013). For to the V. parviflora MSY, but to a different chromosome in example, different sex chromosomes evolved independently V. parviflora.Wealsoexaminedthelocationofthe5Sribo- among several closely related fish species (Woram et al. somal RNA (rDNA) in V. parviflora.Asingle5SrDNA 2003; Takehana et al. 2007, 2008). Sex chromosomes have locus was present on a pair of V. parviflora autosomes. No been best studied in the medaka fish Oryzias latipes (Matsuda 5S rDNA sequences were detected in the V. parviflora MSY et al. 2002, 2007). Interestingly, the sex-determining gene in

Sex Chromosomes in V. parviflora 417 Figure 6 FISH mapping of papaya BACs on V. parviflora X/Y chromosomes. (A) FISH signals derived from five papaya BACs and a telomeric DNA probe on V. parviflora X/Y chromosomes. BAC 5 is located at the short arm boundary of the loop. BAC 6 is located in the middle of the loop. The signals from BAC 6 on the X and Y chromosomes are separated and are indicated by a double arrow. (B) Chromosomes in A were converted into a black-and-white image to enhance the heterochromatic features. (C) FISH signals derived from BACs 21 and 22. These two BACs are located at the distal end of papaya X/Y chromosomes (Figure 5). BAC 22, however, is located in the middle of the V. parviflora X/Y chromosomes. (D) Chromosomes in C were converted into a black-and-white image. (E) FISH signals derived from BACs 16 and 17, which span K3. (F) Chromosomes in E were converted into a black-and- white image. (G) FISH signals derived from BACs 8 and 9. These two BACs are located within the MSY of papaya X/Y chromosomes (Figure 5), but are located at the distal end of the long arm of V. parviflora X/Y chromosomes. (H) Chromosomes in G were converted into a black-and-white image. Unambiguously identified heterochromatic blocks and knobs are marked by blue arrows or arrowheads in B, D, F, and H. Bars, 10 mm.

O. latipes, DMY, was not detected in a sister species O. luzonensis. papaya. An interesting question regarding the results of the Instead, a novel sex-determining gene located on a different current study is whether the X/Y chromosomes in papaya chromosome may have arisen in O. luzonensis and replaced and V. parviflora evolved independently from the same pair DMY (Tanaka et al. 2007). Similarly, S. latifolia is a well-studied of autosomes or from the same pair of ancestral X/Y chro- model plant with X/Y chromosomes (Vyskot and Hobza 2004). mosomes. Since the papaya X/Y chromosomes are estimated S. colpophylla, a species closely related to S. latifolia,contains to have emerged approximately 7 MYA (Yu et al. 2008; sex chromosomes evolved from a different pair of autosomes Wang et al. 2012) and the Vasconcellea/Jacaratia species (Mrackova et al. 2008). are estimated to have diverged from the papaya clade 27 In this study, we demonstrated that V. parviflora contains MYA (Carvalho and Renner 2012), the sex chromosomes in a pair of X/Y chromosomes that are homologous to those of papaya and V. parviflora may have originated independently

418 M. Iovene et al. Figure 7 FISH mapping in J. spinosa. (A) FISH map- ping of BACs 3 and 11. Chromosomal locations of these two clones on the J. spinosa chromosome are similar to those on V. parviflora X/Y chromosomes. (B) Chromosomes in A were converted into a black-and- white image. The J. spinosa chromosomes are homo- morphic and show a complete chromosome pairing. (C) FISH mapping of BACs 3 and 10. Chromosomal locations of these two clones on the J. spinosa chro- mosome are similar to those on V. parviflora X/Y chro- mosomes. (D) Chromosomes in C were converted into a black-and-white image. The J. spinosa chromosomes are homomorphic and show a complete chromosome pairing.

fromthesamepairofautosomes(Figure8A).However,we An alternative interpretation is that the key sex-deter- must be cautious in drawing such a conclusion because the age mining gene(s) emerged before the divergence of papaya estimation of the papaya sex chromosomes was based on a lim- and V. parviflora (Figure 8B). The similar physical sizes of ited amount of DNA sequences and on the nucleotide substi- the two MSYs suggest that the X/Y chromosomes in papaya tution rates of different plant species (Wang et al. 2012). and V. parviflora may have emerged at a similar time. The Additional sequence evidence and calculations will be required MSYs in the two species are located at a similar position on to support the relative young age of the papaya sex chromo- the two Y chromosomes (Figure 5). Remarkably, compara- somes vs. the divergence time of the Caricaceae species. tive FISH mapping revealed that the two MSYs shared only

Figure 8 Models of Y chromosome evolution in papaya and V. parviflora. (A) Independent evolution model. A sex-determining gene (red bar) located in the centro- meric region emerged independently in papaya and V. parviflora, respectively. The two ancestral Y chromo- somes then evolved into the current Y chromosomes in the two species. (B) Monophyletic evolution model. The two current Y chromosomes in papaya and V. parviflora evolved from the same ancestral Y chromosome.

Sex Chromosomes in V. parviflora 419 a small chromosomal domain that is marked by BAC 6 and is work was supported by National Science Foundation Plant close to the centromere of the two Y chromosomes (Figure 5). Genome Research Program awards DBI0553417 and Thus, the sex-determining gene(s) in papaya and V. parviflora DBI0922545. are likely located either within the centromere or closely flanking the centromere of the Y chromosomes. Active genes were reported in the centromeres of plant chromosomes, in- Literature Cited cluding rice (Nagaki et al. 2004; Yan et al. 2006) and potato Bachtrog, D., 2013 Y-chromosome evolution: emerging insights (Gong et al. 2012). In addition, chromosomal crossings over into processes of Y-chromosome degeneration. Nat. Rev. Genet. are completely suppressed within centromeres as well as 14: 113–124. regions immediately flanking the centromeres (Yan et al. Bellott, D. W., H. Skaletsky, T. Pyntikova, E. R. Mardis, T. Graves 2005, 2008). Thus, the location of the newly emerged sex- et al., 2010 Convergent evolution of chicken Z and X determining gene(s) in the centromeric region is favorable for chromosomes by expansion and gene acquisition. Nature 466: 612–616. the survival of these genes during evolution. Carvalho, F. A., and S. S. Renner, 2012 A dated phylogeny of the If the starting position of the two MSYs is around the papaya family (Caricaceae) reveals the crop’s closest relatives centromere, then the papaya MSY has expanded toward the and the family’s biogeographic history. Mol. Phylogenet. Evol. long arm of the chromosome and the V. parviflora MSY to- 65: 46–53. ward the short arm (Figure 5). The differential expansions Charlesworth, D., 2013 Plant sex chromosome evolution. J. Exp. Bot. 64: 405–420. of the two MSYs are also vindicated by several cytogenetic Cheng, Z. K., C. R. Buell, R. A. Wing, and J. M. 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Sex Chromosomes in V. parviflora 421 GENETICS

Supporting Information http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.114.173021/-/DC1

Evidence for Emergence of Sex-Determining Gene(s) in a Centromeric Region in Vasconcellea parviflora

Marina Iovene, Qingyi Yu, Ray Ming, and Jiming Jiang

Copyright © 2015 by the Genetics Society of America DOI: 10.1534/genetics.114.173021 Figure S1. FISH mapping of 5S rDNA on pachytene chromosomes of V. parviflora. (A) FISH using a 5S rDNA probe in a pachytene cell of V. parviflora. The FISH signal from a single 5S rDNA locus is indicated by a red arrow. The loop on X/Y chromosome is indicated by a green arrow. Bar = 10 µm. (B) Chromosomes in (A) were converted into black-white to enhance the heterochromatic features.

2 SI M. Iovene et al. Table S1. List of papaya BAC clones used in comparative FISH mapping BAC BAC V. parviflora(c) Papaya(d) N(e) Location on V. parviflora number(a) name(b) X/Y 1 55G05 13.8 ± 2.0 2.7 ±1.0 18 Short arm 2 19A10 23.0 ± 2.7 21.3 ± 0.8 3 " 3 73P20 23.1 ± 2.9 24.0 ± 1.1 7 " 4 14L22 23.5 ± 1.5 26.6 ± 1.1 4 " 5 19I15 32.4 ± 4.4 39.5 ± 2.9 8 Before B1 86B15 Repetitive Border B* na Pericentromeric signals on multiple chromosomes 6 53E18 38.5 ± 2.2 K5* 8 Between B1 and K1 84J07 Repetitive Between the gap and na Pericentromeric signals on K5** multiple chromosomes 52H15 " K4 on MSY* na " 7 54M13 Not on X/Y Flanking the gap on X ** na End of another chromosome M136D11 Repetitive Flanking the gap on X ** na Pericentromeric signals on multiple chromosomes 69D24 " Between K3 and gap** na " 79M13 " Between K3 and gap** na " 61H02 " K3 – corresponds to the na " BAC right before the gap at border A on HSY* 8 80F18 98.9 ± 0.6 Between K1 and K3** 6 End of the long arm 9 93K15 96.7 ± 0.5 Between K1 and K3** 9 End of the long arm 10 54M22 95.8 ± 0.9 Between K1 and K3** " End of the long arm; additional signal on another 84M10 95.8 ± 0.9 Border A** 7 Overlaps to 54M22; additional signal on a knob of another 19B15 98.9 ± 0.6 51.7 ± 1.9*** 6 Partially overlaps with 80F18 13 01M23 89.9 ± 1.7 67.4 ± 2.6 6 Long arm 14 88M12 84.5 ± 2.1 74.4 ± 2.5 6 " 15 86F03 62.0 ± 4.2 77.2 ± 2.8 6 " 16 04E12 66.2 ± 1.5 79.6 ± 2.7 3 " 17 13I21 70.5 ± 2.5 83.6 ± 3.0 3 " 18 26N04 71.0 ± 1.3 86.5 ± 3.1 3 " 19 19E20 77.4 ± 2.8 Between BAC 18 and 6 " BAC 20 K3 Knob 67.6 ± 4.3 Between BAC 19 and 15 Between BAC 16 and BAC BAC 20 17 20 96C17 81.9 ± 1.4 95.0 ± 1.5 10 Long arm 21 90J10 " 96.2 ± 1.0 Partially overlaps with 96C17 22 99B02 55.0 ± 3.7 98.5 ± 1.1 15 Partially overlaps with K2 (a) Only the clones with distinctive hybridization signal in both papaya and V. parviflora are numbered. (b) BAC clones marked in red bold are specific to the papaya X chromosome, BAC 52H15 in blue bold is Y specific. (c) Relative physical position: (D/L) x 100, where D is the distance (in micrometers) from the FISH site to the end of the short arm of the chromosome, and L is the total length of the chromosome (in micrometers). (d) Relative physical positions for papaya are according to: Wai et al. (2012, Chromosome Res. 20: 753-767); (*) Zhang et al. (2008, Genome Res. 18: 1938-1943); (**) Na et al. (2012, BMC Genomics 13: 176). (***) FISH position of BAC 12 relative to BAC 11 is unknown in papaya. (e) Number of measurements. na: not available.

3 SI M. Iovene et al. Table S2. Summary of chromosomal positions of papaya BACs in V. parviflora and J. spinosa BAC BAC V. parviflora J. spinosa number1 name 3 73P20 Middle of the short arm, similar Middle of the short arm, similar to papaya to papaya 5 19I15 Interstitial, similar to papaya Interstitial, similar to papaya 6 53E18 Interstitial, similar to papaya Interstitial, similar to papaya 7 54M13 End of a different chromosome End of a different 80F18 Chromosome end opposite to Chromosome end opposite to 73P20, different from papaya 73P20, different from papaya 9 93K15 Chromosome end opposite to Chromosome end opposite to 73P20, different from papaya 73P20, different from papaya 10 54M22 Chromosome end opposite to Chromosome end opposite to 73P20; another signal on a 73P20; another signal on a different chromosome different chromosome 11 84M10 Chromosome end opposite to Chromosome end opposite to 73P20; second signal on another 73P20; no second signal chromosome 12 19B15 Chromosome end opposite to Chromosome end opposite to 73P20 73P20 1 The chromosomal positions of the clones in bold are different from papaya.

4 SI M. Iovene et al.