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–Instituteof 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 ofIllinois 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 studythe evolutionary histories of sex chromosomes. We previously demonstrated that papaya (Carica papaya) (2n = 2x = 18), a fruit tree in thefamily Caricaceae, contains recently emerged but cytologically heteromorphic X/Y chromosomes. We have been intrigued by the possiblepresence 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 ofVasconcellea parviflora (2n = 2x = 18), a species that diverged from papaya �27 million years ago. We demonstrate that V. parvifloracontains a pair of heteromorphic X/Y chromosomes that are homologous to the papaya X/Y chromosomes. The comparative mapping resultsrevealed that the male-specific regions of the Y chromosomes (MSYs) probably initiated near the centromere of the Y chromosomes in bothspecies. The two MSYs, however, shared only a small chromosomal domain near the centromere in otherwise rearranged chromosomes. TheV. parviflora MSY expanded toward the short arm of the chromosome, whereas the papaya MSY expanded in the opposite direction. MostBACs mapped to papaya MSY were not located in V. parviflora MSY, revealing different DNA compositions in the two MSYs. These resultssuggest 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

HETEROMORPHIC sex chromosomes (X/Y, Z/W) evolvedfrom pairs of autosomes (Ming et al. 2011; Bachtrog

2013; Charlesworth 2013). It is generally accepted that sexchromosome evolution is initiated by the emergence of a sex-determining locus. Suppression of recombination surround-ing the sex-determining locus, which favored the linkage ofthe sex-determining alleles with sexually antagonistic alleles,caused the decay of the Y/W chromosomes and resulted ina pair of heteromorphic sex chromosomes (Ming et al. 2011;Bachtrog 2013; Charlesworth 2013). Sex chromosomes evolvedindependently in different eukaryotic lineages. The mammalianX chromosome and the bird Z chromosome evolved from dif-ferent portions of the ancestral genome (Bellott et al. 2010;Cortez et al. 2014). Interestingly, all therian mammals have

homologous XY systems and all birds have homologous ZWsystems (Graves 2008). Thus, these sex chromosomes, sur-prisingly, appear to have evolved only once in each of thesetwo major evolutionary clades.

Compared to mammals and birds, sex chromosomes haveemerged more frequently in several other eukaryotic lineages,including plants. Sex chromosomes have been reported in atleast 48 plant species across 20 different families, includingboth X/Y and Z/W systems (Ming et al. 2011; Kumar et al.2014). Sex chromosomes in some plant species were identi-fied using genetic mapping-based approaches. However, cyto-genetic data are not available from most of these species. Thus,it is unclear whether heteromorphic chromosomes have al-ready developed in these species, such as asparagus (Asparagusofficinalis) (Telgmann-Rauber et al. 2007), Populus (Populustrichocarpa) (Yin et al. 2008), and spinach (Spinacia oleracea)(Yamamoto et al. 2014). Heteromorphic sex chromosomeswere revealed cytologically in several plant species (Yamatoet al. 2007; Zhang et al. 2008; Sousa et al. 2013), including,Silene latifolia, which is one of the best-studied model plantspecies with well-differentiated X/Y chromosomes (Vyskot

Copyright © 2015 by the Genetics Society of Americadoi: 10.1534/genetics.114.173021Manuscript received October 4, 2014; accepted for publication November 26, 2014;published Early Online December 5, 2014.Supporting information is available online at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.114.173021/-/DC1.1Corresponding author: Department of Horticulture, 1575 Linden Dr., Room 409A,University of Wisconsin, Madison, WI 53706. E-mail: jjiang1@wisc.edu

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 plantspecies (Hizume et al. 1988; Howell et al. 2009; Mariottiet al. 2009; Navajas-Perez et al. 2009). Most of the plantspecies with sex chromosomes have not been sequenced.We have limited knowledge of the evolutionary histories ofplant sex chromosomes because of scarce genomic resourcesand 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 statein 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 fewmillion years ago (MYA) (Yu et al. 2008; Wang et al. 2012).The young nature of papaya sex chromosomes was confirmedcytologically in which only a small portion (�13%) of the X/Ychromosomes is cytologically differentiated (Zhang et al. 2008).Thus, papaya provides a model system to capture early molec-ular and cytological events during sex chromosome evolution.The Vasconcellea/Jacaratia clade in Caricaceae is closely relatedto papaya (Carvalho and Renner 2012) (Figure 1). We wereintrigued to know whether the dioecious Vasconcellea/Jacaratiaspecies contains sex chromosomes and whether such sex chro-mosomes are related to the papaya X/Y chromosome. We con-ducted fluorescence in situ hybridization (FISH) of a set ofbacterial artificial chromosome (BAC) clones, all of whichwere previously mapped to the papaya X/Y chromosomes, intwo Caricaceae species, Vasconcellea parviflora (2n= 2x= 18)and Jacaratia spinosa (2n = 2x = 18). We demonstrate thatV. parviflora contains a pair of heteromorphic X/Y chromo-somes that are homologous to the papaya X/Y chromosomes.By contrast, the same pair of chromosomes in J. spinosa ishomomorphic. These results reveal a dynamic nature of sexchromosome evolution in the Caricaceae species.

Materials and Methods

Materials

Plants of V. parviflora and J. spinosa were maintained at theHawaii Agriculture Research Center as well as in the TexasAgriLife Research Center in Weslaco, Texas. Young maleflower buds were collected and fixed in 3:1 (100% ethanol:glacial acetic acid) Carnoy’s solution and kept at 220� untiluse. Twenty-nine papaya BAC clones were previously selectedand were either specific to the papaya sex-determining region(X or Y specific) or to the papaya sex chromosomes (Zhanget al. 2008; Wai et al. 2012). A telomeric DNA probe, pAtT4from Arabidopsis thaliana (Richards and Ausubel 1988), wasused to label the ends of V. parviflora pachytene chromosomes.

FISH

Chromosome preparation and FISH followed previouslypublished protocols (Cheng et al. 2002; Iovene et al.2008). BAC DNA was labeled with either biotin-16-dUTP

or digoxigenin-11-dUTP (Roche Diagnostics, Indianapolis, IN),using a standard nick translation reaction. Chromosomes werecounterstained with 49,6-diamidino-2-phenylindole (DAPI) inVectaShield antifade solution (Vector Laboratories, Burlingame,CA). The FISH images were processed with Meta Imaging Se-ries 7.5 software. The final contrast of the images was processedusing Adobe Photoshop CS3 software. Heterochromatin lengthwas estimated based on DAPI staining pattern of the putative Ychromosome and expressed in percentage of the total chromo-some length. The Y-specific heterochromatic domain was esti-mated as the difference in heterochromatin content betweenX and Y chromosomes and was expressed as the percentageof the total chromosome length. The position of a BAC cloneor other landmarks along the chromosome was estimated andexpressed as D/L3 100, where D= distance of the landmarkfrom the end of the short arm, and L = total chromosomelength. All measurements were made on digital photographs,using Meta Imaging Series 7.5 software.

Results

Identification of the X/Y chromosomes in V. parviflora

V. parviflora is a dioecious species with female and maleindividuals. Both male and female flowers are morphologi-cally similar to those of papaya (Figure 2). Young flowerbuds were collected from male V. parviflora plants to preparemeiotic pachytene chromosomes. The pachytene karyotype ofV. parviflora generally resembled the papaya karyotype (Zhanget al. 2010), containing nine metacentric and submetacentricchromosomes of similar sizes (Figure 3C). Heterochromatin,which is brightly stained by DAPI, was visible in the pericentro-meric regions of all nine pachytene chromosomes (Figure 3C).Surprisingly, a large loop was consistently observed on one ofthe pachytene chromosomes (Figure 3A). The two homologouschromosomes were separated and unpaired within the loop.

Figure 1 Phylogenetic relationships in family Caricaceae. Branch lengthsare proportional to time. Numbers on nodes represent age in millionyears. This chronogram is adapted from Carvalho and Renner (2012).Species sampled in this study are in boldface type.

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A large and a small heterochromatic domain were observed ononly one of the two homologs in the loop (Figure 3B).

We next conducted FISH analysis on V. parviflora pachy-tene cells, using several BAC clones that were previouslymapped to the papaya X/Y chromosomes. Most of theseBACs were mapped to this loop-associated V. parviflorachromosome pair, suggesting that they are homologous tothe papaya X/Y chromosomes (Figure 3C). Therefore, thispachytene chromosome pair is likely the sex chromosomepair in V. parviflora and the loop region represents theMSY. Significant accumulation of heterochromatin and pos-sibly chromosomal rearrangements, such as inversions and/or duplications, within the loop may prevent pairing of theputative X and Y chromosomes in this region.

Distinct heterochromatin distribution patternsassociated with the V. parviflora X/Y chromosomes

We prepared pachytene chromosomes at both early and latestages to further investigate chromatin differentiation betweenthe X and Y chromosomes. Two large heterochromatin blocks(B), B1 and B2, and three small heterochromatic knobs (K),K1, K2, and K3, were observed on the X/Y chromosomes(Figure 4). These heterochromatic domains were best visual-ized on middle pachytene chromosomes (Figure 4B). B1 andK1 were observed only on one of the two homologs, mostlikely the Y chromosome. B1 was consistently located in theloop on both early and late pachytene chromosomes. K1 wasmuch smaller than B1, but was detectable within the loop onmost pachytene chromosomes. Heterochromatic domainswere often divided into two or more separate subdomainson highly extended early pachytene chromosomes (Figure 4A).

The heterochromatin distribution pattern on V. parvifloraX/Y chromosomes is similar to the pattern observed on papayaX/Y chromosomes. The papaya X/Y pachytene chromosome

contains five knobs, including one large knob shared by Xand Y and four small knobs specific to the papaya MSY (Zhanget al. 2008). The loop associated with the V. parviflora X/Ychromosomes is located at a similar position to that of the MSYon the papaya Y chromosome (Figure 5). The length of theloop accounted for 12% (11.7 6 3.04, n = 15) of theV. parviflora Y chromosome. In comparison, the MSY of pa-paya accounts for 13% of the papaya Y chromosome (Zhanget al. 2008). However, more heterochromatin has accumu-lated in the V. parviflora MSY than in the papaya MSY. Noloop was observed in the papaya MSY, although the Y chro-mosome, which contains 8.1 Mb DNA compared to 3.5 Mb forthe corresponding X region (Wang et al. 2012), has to twist(zigzag) in pairing with the X within the MSY (Zhang et al.2008). In comparison, the V. parviflora Y chromosomeappeared to have accumulated much more DNA than the Xchromosome in the loop. Self “pairing” or “supercoiling” ofthe Y chromosome was observed within the loop on earlypachytene chromosomes (Figure 4A).

The centromere of the papaya Y chromosome was mappedclose to knob 4 within the MSY (Zhang et al. 2008) (Figure5). The chromosomal domain between B1 and K1 in theV. parviflora MSY was consistently less stained and appearedmorphologically to be the primary constriction. Based on thisstructure, as well as the synteny between papaya X/Y andV. parviflora X/Y chromosomes (see below), the centromereof the V. parviflora Y chromosome is predicted to be locatedbetween B1 and K1 within the loop (Figure 5).

Comparative FISH mapping of the X/Y chromosomes inpapaya and V. parviflora

We selected a total of 29 BAC clones that were previouslymapped to the papaya X/Y chromosomes (Zhang et al. 2008;Wai et al. 2010; Na et al. 2012), including 11 BACs specific

Figure 2 Morphology of flowers from papaya andV. parviflora. (A) Papaya male flowers. (B) Papayafemale flowers, which occur individually or in clus-ters. (C) V. parviflora male flowers. (D) V. parviflorafemale inflorescence, which usually contains .10flowers.

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to X, 1 BAC specific to Y, and 17 BACs shared by X and Y(Supporting Information, Table S1). These BACs were map-ped to the homologous V. parviflora chromosomes by FISHto further examine the syntenic relationship of the sex chro-mosomes in the two species. Only 22 papaya BACs gener-ated distinct FISH signals on V. parviflora chromosomes.These clones were labeled in numbers from 1 to 22 (Figure 5,Table S1).

Five BACs, including clones 8–11 that are located in thepapaya MSY, were surprisingly mapped to the distal regionon the long arm of V. parviflora X/Y chromosomes (Figure 5).By contrast, BAC 22, the most distal clone on the long arm ofpapaya X/Y chromosomes, was mapped to the middle of theV. parviflora X/Y chromosomes (Figure 5 and Figure 6C).Thus, an inversion (inversion 1 in Figure 5), which spans

almost the entire long arm, occurred in one of the twospecies after divergence from a common ancestor. The re-verse order of BACs 13 and 14 in the two species (Figure 5)also supports this inversion hypothesis. Interestingly, BACs15–21 maintained the same order on the long arm in thetwo species (Figure 5 and Figure 6E). Thus, an indepen-dent and smaller inversion (inversion 2 in Figure 5) spanningBACs 15–21 occurred within the chromosomal domainspanned by inversion 1.

BACs 1–6, which span the short arm of papaya X/Y chro-mosomes, maintained the same order on V. parviflora X/Ychromosomes (Figure 5 and Figure 6A). However, the dis-tance between BAC 1 and the telomere was much shorter inpapaya than in V. parviflora, suggesting another inversion inthis region (inversion 3 in Figure 5).

Figure 3 Pachytene chromosomes of V. parviflora. (A)A complete pachytene cell of V. parviflora. The greenFISH 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 chromosomepair is included in a white box. Bar, 10 mm. (B) TheDAPI-stained chromosomes in A were converted intoa black-and-white image to enhance the visualizationof heterochromatin. The X (red) and Y (green) chro-mosomes within the loop are illustrated in the inset. Alarge Y-specific heterochromatin domain in the loop isillustrated in blue. (C) A pachytene karyotype devel-oped from the same cell as in A. Individual chromosomeswere digitally isolated, straightened, and arranged basedon their length, except that the X/Y chromosome pairwas arranged as the first. The arrow points to the FISHsignal derived from BAC 1.

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

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Comparative FISH mapping of the MSYs in papaya andV. parviflora

Although the two MSYs shared a similar chromosomal locationand size, they differed in structure and DNA composition. Thechromosomal domain spanned by BACs 8–11 represented a sig-nificant part of the papaya MSY. This region, however, waslocated at the distal end of the long arm of V. parviflora X/Ychromosomes (Figure 5). In addition, BAC 7 was not mappedto the V. parviflora MSY, but to a different chromosome inV. parviflora. We also examined the location of the 5S ribo-somal RNA genes (rDNA) in V. parviflora. A single 5S rDNAlocus was present on a pair of V. parviflora autosomes. No5S rDNA sequences were detected in the V. parviflora MSY

(Figure S1). By contrast, massive amounts of 5S rDNA-relatedsequences were amplified in the papaya MSY (Zhang et al.2010).

Based on the position of the Y chromosome centromerewithin the MSY, the majority of the papaya MSY is locatedon the long arm of the Y chromosome (Zhang et al. 2008). Incontrast, the heterochromatic domain B1 represents morethan half of the V. parviflora MSY (Figure 4). This region,spanned by BACs 5 and 6, was significantly expanded inV. parviflora compared to the same region in papaya (Figure5). Since the centromere of the V. parviflora Y chromosomeis located between K1 and B1, the majority of the V. parvifloraMSY is located on the short arm of the Y chromosome (Figure5). Thus, the papaya MSY has expanded toward the long armof the Y chromosome, whereas the V. parviflora MSY hasexpanded toward the short arm of the Y chromosome.

J. spinosa chromosomes homologous to papaya X/Ychromosomes are homomorphic

Jacaratia is one of six genera in Caricaceae. Phylogeneticanalysis revealed that Jacaratia and Vasconcellea speciesare equally distant from papaya (Carvalho and Renner2012) (Figure 1). We were interested whether Jacaratiaspecies also shared the same sex chromosomes with papayaand V. parviflora. We chose one dioecious species, J. spinosa(2n = 2x = 18), for comparative FISH mapping.

We conducted FISH analysis, using nine papaya BACclones (BACs 3 and 5–12) (Table S2) on J. spinosa chro-mosomes. The chromosomal positions of all nine BACs inJ. spinosa were similar to those observed on V. parvifloraX/Y chromosomes, including the distal locations of BACs8–12 (Figure 7 and Table S2). Therefore, the three inversionsassociated with this chromosome in Caricaceae species arenot specific to V. parviflora and may have emerged duringthe evolution of the papaya genome.

Surprisingly, the J. spinosa chromosome pair that is ho-mologous to the papaya/V. parviflora X/Y chromosomesshowed perfect pairing at the pachytene stage without anyloop. No chromatin differentiation between the two homo-logs was observed on this pachytene chromosome (Figure 7,B and D). We did not observe any heteromorphic pachytenechromosome in J. spinosa. However, it is not conclusive re-garding the absence of heteromorphic sex chromosomes inthis species because we were able to collect and analyze onlyfew meiotic cells at the pachytene stage.

Discussion

Sex chromosomes emerged multiple times independently inseveral eukaryotic lineages, including fishes, reptiles, andplants (Graves 2008; Ming et al. 2011; Bachtrog 2013). Forexample, different sex chromosomes evolved independentlyamong several closely related fish species (Woram et al.2003; Takehana et al. 2007, 2008). Sex chromosomes havebeen best studied in the medaka fish Oryzias latipes (Matsudaet al. 2002, 2007). Interestingly, the sex-determining gene in

Figure 5 Comparative mapping of the X/Y chromosomes in papaya(adapted from Zhang et al. 2010; Wai et al. 2012) and V. parviflora. BACsin red numbers in boldface type are specific to the papaya X chromo-some. BAC 11* generated signals on an additional V. parviflora chromo-some. BAC 7** was mapped to a different chromosome in V. parviflora.The green dashed lines mark the putative positions of the centromeres onthe Y chromosomes. The position of the papaya MSY and the loop onV. parviflora Y chromosomes are marked. Three inversions are predictedbased on the order and position of BAC clones in the two species. Thedifferent locations of BAC 1 in the two species are illustrated by a blackdashed line. Blue and red dashed lines highlight the centromeric vs.telomeric positions of the four BACs in the two species. Note that thethree inversions drawn along the V. parviflora Y chromosome likelyemerged during the evolution of papaya.

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O. latipes, DMY, was not detected in a sister species O. luzonensis.Instead, a novel sex-determining gene located on a differentchromosome may have arisen in O. luzonensis and replacedDMY (Tanaka et al. 2007). Similarly, S. latifolia is a well-studiedmodel plant with X/Y chromosomes (Vyskot and Hobza 2004).S. colpophylla, a species closely related to S. latifolia, containssex chromosomes evolved from a different pair of autosomes(Mrackova et al. 2008).

In this study, we demonstrated that V. parviflora containsa pair of X/Y chromosomes that are homologous to those of

papaya. An interesting question regarding the results of thecurrent study is whether the X/Y chromosomes in papayaand V. parviflora evolved independently from the same pairof autosomes or from the same pair of ancestral X/Y chro-mosomes. Since the papaya X/Y chromosomes are estimatedto have emerged approximately 7 MYA (Yu et al. 2008;Wang et al. 2012) and the Vasconcellea/Jacaratia speciesare estimated to have diverged from the papaya clade �27MYA (Carvalho and Renner 2012), the sex chromosomes inpapaya and V. parviflora may have originated independently

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 probeon 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 fromBAC 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-whiteimage to enhance the heterochromatic features. (C) FISH signals derived from BACs 21 and 22. These two BACs are located at the distal end of papayaX/Y chromosomes (Figure 5). BAC 22, however, is located in the middle of the V. parviflora X/Y chromosomes. (D) Chromosomes in C were convertedinto 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 arelocated 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.

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from the same pair of autosomes (Figure 8A). However, wemust be cautious in drawing such a conclusion because the ageestimation of the papaya sex chromosomes was based on a lim-ited amount of DNA sequences and on the nucleotide substi-tution rates of different plant species (Wang et al. 2012).Additional sequence evidence and calculations will be requiredto support the relative young age of the papaya sex chromo-somes vs. the divergence time of the Caricaceae species.

An alternative interpretation is that the key sex-deter-mining gene(s) emerged before the divergence of papayaand V. parviflora (Figure 8B). The similar physical sizes ofthe two MSYs suggest that the X/Y chromosomes in papayaand V. parviflora may have emerged at a similar time. TheMSYs in the two species are located at a similar position onthe two Y chromosomes (Figure 5). Remarkably, compara-tive FISH mapping revealed that the two MSYs shared only

Figure 7 FISH mapping in J. spinosa. (A) FISH map-ping of BACs 3 and 11. Chromosomal locations ofthese two clones on the J. spinosa chromosome aresimilar 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. Chromosomallocations 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 intoa black-and-white image. The J. spinosa chromosomesare homomorphic and show a complete chromosomepairing.

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

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a small chromosomal domain that is marked by BAC 6 and isclose to the centromere of the two Y chromosomes (Figure 5).Thus, the sex-determining gene(s) in papaya and V. parvifloraare likely located either within the centromere or closelyflanking the centromere of the Y chromosomes. Active geneswere reported in the centromeres of plant chromosomes, in-cluding rice (Nagaki et al. 2004; Yan et al. 2006) and potato(Gong et al. 2012). In addition, chromosomal crossings overare completely suppressed within centromeres as well asregions immediately flanking the centromeres (Yan et al.2005, 2008). Thus, the location of the newly emerged sex-determining gene(s) in the centromeric region is favorable forthe survival of these genes during evolution.

If the starting position of the two MSYs is around thecentromere, then the papaya MSY has expanded toward thelong arm of the chromosome and the V. parviflora MSY to-ward the short arm (Figure 5). The differential expansionsof the two MSYs are also vindicated by several cytogeneticmapping results:

1. The V. parviflora MSY is more heterochromatic than thepapaya MSY. The V. parviflora MSY is consistently asso-ciated with an unpaired chromosomal loop, whereas theX and Y chromosomes within papaya MSY can pair, al-though in an irregular fashion (Zhang et al. 2008).

2. The papaya MSY has accumulated massive amounts ofrepetitive DNA sequences derived from 5S ribosomal RNAgenes (Zhang et al. 2010). By contrast, we did not detectany 5S rDNA-related DNA sequences in V. parviflora MSY(Figure S1).

3. Several BACs showed different hybridization patterns inthe two MSYs. BAC 7 is associated with papaya MSY, butit hybridizes to a different chromosome in V. parviflora.BAC 6 is specific to X in papaya, but it hybridizes to bothX and Y in V. parviflora.

J. spinosa contains a pair of chromosomes that sharea perfect synteny with the V. parviflora X/Y chromosomes(Table S2). Interestingly, no loop or chromatin differentiationwas observed on this pair of J. spinosa chromosomes at thepachytene stage. These results can be interpreted by two hy-potheses: (1) Sex determination in J. spinosa is associated withthe same pair of chromosomes as papaya and V. parviflora, butthe primitive X/Y chromosomes in J. spinosa have not differ-entiated cytologically and appear to be homomorphic, and (2)a different pair of chromosomes is associated with sex deter-mination in J. spinosa. Thus, J. spinosa provides an excel-lent model for future study of sex chromosome evolution inCaricaceae species.

Acknowledgments

We thank Fernanda Carvalho for help on the developmentof Figure 1 and Li He for development of Figure 3C in themanuscript. We are grateful to Francis Zee, Jim Carr, andother members of the Zee laboratory for help in the collectionof meiotic samples of Vasconcellea/Jacaratia species. This

work was supported by National Science Foundation PlantGenome Research Program awards DBI0553417 andDBI0922545.

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Communicating editor: D. A. Barbash

Sex Chromosomes in V. parviflora 421

GENETICSSupporting 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 AmericaDOI: 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.

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Table S1. List of papaya BAC clones used in comparative FISH mapping BAC

number(a) BAC

name(b) V. parviflora(c) Papaya(d) N(e) Location on V. parviflora

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

K5** na Pericentromeric signals on

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

BAC right before the gap at border A on HSY*

na "

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 chromosome

11 84M10 95.8 ± 0.9 Border A** 7 Overlaps to 54M22; additional signal on a knob of another chromosome

12 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

BAC 20 6 "

K3 Knob 67.6 ± 4.3 Between BAC 19 and BAC 20

15 Between BAC 16 and BAC 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.

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Table S2. Summary of chromosomal positions of papaya BACs in V. parviflora and J. spinosa BAC

number1 BAC name

V. parviflora J. spinosa

3 73P20 Middle of the short arm, similar to papaya

Middle of the short arm, similar 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 chromosome 8 80F18 Chromosome end opposite to

73P20, different from papaya Chromosome end opposite to 73P20, different from papaya

9 93K15 Chromosome end opposite to 73P20, different from papaya

Chromosome end opposite to 73P20, different from papaya

10 54M22 Chromosome end opposite to 73P20; another signal on a different chromosome

Chromosome end opposite to 73P20; another signal on a different chromosome

11 84M10 Chromosome end opposite to 73P20; second signal on another chromosome

Chromosome end opposite to 73P20; no second signal

12 19B15 Chromosome end opposite to 73P20

Chromosome end opposite to 73P20

1 The chromosomal positions of the clones in bold are different from papaya.

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