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  1. 1. Gonutics: Plbflsllad Articles Ahead ot Plilt, published on July 1. 2007 as 10.1534lgonot| cs.107.071175 Early events In the evolution ol the Sllene lanifolla Y chromosome: male speelallzntlon and recombination arrest lilka Zluvm'a"'. Scvdalin Gcorgicv‘. Bohuslav Janouck‘. Deborah Char| cswnnh', Boris Vysknf, loan Ncgnutiu’ ‘ lmboratnry of Plum Drrclnpmenml (icnelicx. lnxlitule of Bioph_'. n’c. ‘ Qf the Academy of Science: of the C zeth Republic 1'. v. i. . 612 65 Brno. C zed! Republic ’ L’m‘versiIe' de Lyon. ENS Lyon. Inbomloirz RDP, IF RI 28 BiaSrienres L_mn-Garland. 69364 Lyon. Franre ‘Faculty ufBt'olog_'. Drparlmenl of Genetics. Sofia L/ nirrrsily. I I64 Sofia. Bulgaria " Inslitule af Evululitmary Biology, L’n1'w'r3it_v of Edinburgh. King '3 Buildings; Wen Main: Road. Edinburgh. EH9 JJT. UK
  2. 2. Silt-ne huifolia male function genes. p. 2 Running title: Silent Iarifnlia male function genes Key words: Silcne laufalia, Y chromosome, male—indispensable genes. X-Y paring contml. deletion mapping Corresponding author: loan Ncgrutiu. Université dc Lynn. ENS Lyon. Lahormnire RDP, lFRl28 Bioscienccs Lynn-Gerlnnd. 46All6e d'ltalic. 69364 Lyon. France. E-mail: lunn. Ncgruuu @cns-lyon. fr Phone: 0033 4 72 72 86 I2
  3. 3. Silent» lau'j’olia male function genes. p. 3 ABSTRACT Understanding the origin and evolution of sex chromosomes requires studying recently evolved X-Y chromosome systems such as those in some flowering plants. We describe Y chromosome deletion mutants of Silt! !! latt'fnlt'a. a dioeciouw plant with hctcmrnorphic sex chmmosorncs. Combination of the results from new and previously described deletions with histological descriptions of their statnen development defects indicates the presence of two distinct Y regions containing loci with it-tdiapcnsable roles in male reproduction. We determine their positions relative to the two main sex rlelerrnination function (female suppressing and the other male pmrnotirtg). A region proximal to the centrurnete on the Y p arm containing the putative stumen promoting sex determination locus includes additional early stamen developmental factors. A media] region of the Y q amt carries late pollen fertility factors. Cytological analysis of rneiotic X-Y pairing in one of the male-sterile mutants indicates that the Y carries sequences or functions specifically affecting sex chromosome pairing.
  4. 4. Slime Iatifolia male function genes. p. 4 INTRODUCTION It is widely accepted that X-Y sex chromosome systems evolved from a pair of autosomes. which initially acquired the mntationls) respomihle for sexual dimorphism. the "primary" sex determination genes (reviewed in CHARLESWORTH l99I). Two main processes an: generally considered to occur during this earliest stage of X-Y evolution. One is restriction of crossing-over in the nzgionts) containing these loci. which might be called a proto— X-Y system. The second is the accumulation of two kinds of genes advantageous for male sex functions. sexually antagonistic male benefit alleles. and alleles enhancing male gamete success. Sexually antagonistic male benefit alleles are predicted to accumulate close to the sex determining genes. because exprtssion of their disadvantageous effects in females will be reduced by tight linkage with the sex determining region (HSHFJI I931: RICE I987; reviewed in Vtcoso and CllAltL£SWOR1’ll 2006). Recombination between the primary sex-determining region and loci at which rnale-benefit alleles have accumulated on the Y will generate poorly adapted phenotypes. selecting for tighter linkage in the region. The process of selection and recruitment of male-benefit alleles may be followed by further selection favoring restriction of recombination. which may eventually be completely suppressed. and the entire “pmto-Y- chromosome” region may become non-recombining. This can explain why. in many sex chromosome systerm. recombination is suppressed across most of the Y. not just in the primary sex-determining region (reviewed in CHAttLt= .sw0It‘rH at al. 2tIl5). In mammals. for exzunple. only small parts of the Y (the “pseudo-autuaoninl" PARI and PAR2: see PERRY and Asnwonm I99‘) and CHA. RCHAR er al. 2003) still recombine with the X. After crossing-over ceases. the sequences of the initially homologous regiom of the proto-sex chromosomes diverge. both due to funlter mutations in genes linked to the sex- dctermining region. to just outlined. and to substitutions of neutral and weakly dclcteriots
  5. 5. Silrne latij’olia male function genes. p. 5 mutations under genetic drift. The low effective population size of the evolving Y chromosome also leads to genetic degeneration within the non-recombining sex-specific region. including rearction of genes’ functions. loss of genes, and accumulation of rranspmahle elements (reviewed in CttMtt. t=. swott1'H and CHArtt. t=5wotrrH 2000). Empirical evidence for the involvement of sexually antagonistic male benefit alleles, and for their presence on the Y driving the evolution of low crossing-over, is limited. The classical evidence for accurnulation of male benefit alleles closely linked to sex-deterrninirrg genes is the presence, in the fish Poecilia reticulum. of male sexual attractiveness alleles in both the non-recombining and pseudo-autosomal regions of the Y chromosome (WINGE I927: LINDHOLM and BREDEN 2002; BULL I983). These alleles reduce males’ survival to maturity. even under laboratory conditions (BROOKS 2000). so they are clearly disadvantageous. except for their effects on male sexual attractiveness. They are probably not sex-limited in expression (BULL I983). but the benefits of attractiveness in females are less important than for males. and there are fitness costs of ornamentation in terms of conspicuousness to predators: thus these are probably sexually antagonistic genes of the kind hypothesized. There is also some quantitative genetic support for sexually antagonistic alleles in Dmropltila melanngarrer (GIBSON er al. 2002). and there is indirect evidence in the neo-Y system of D. americwta. (MCALUSTBR and EVANS 2(X)6). Recently, a QT L mapping surely inferred the presence of sexually antagonistic genes for sexually dimorphic characters in S. laltfolin. including flower size (Scam and DELPH 2006). However. these conclusions need to be verified by other kinds of txurrlters. as the genes were inferred to be located in two PAR regions (which has not previously been sumeeted). Moreover. the situation is not the one hypothesized above: females apparently do not express the phenotypes (expression is sex-lirnitetl). and the genes were hypothesized to be maintained polymorphic by antagonistic pleiotropy in males. An indirect line of evidence is based on the human Y. the best studied Y chromosome so far. As the theory above predicts. the Y sequence has revealed numerous genes involved in
  6. 6. Silrnr latifnlia male function genes. p. 6 spcrmatogcncsis. Some of these are genes that were once autosotnal or X-linked. and have transposed onto the Y (LAMN and PAGI-. 199919: SKAL| :"l‘SKY el ul. 2003). while others are multi»copy genes. present in "antpliconic regions". mostly with no homology with either X- linked or autosorrtal genes (SI(. »l. I'; TsttY el al. 2003). The human Y is thus enriched for male fttnction genes. many of which are indispensable for male reproduction (their loss leads to oligospenrtirt or azoospermia, disorders which disallow reproduction under natural conditions‘. these data are reviewed in KEhT-l-‘tKs‘r 2(K)0; Hus and AFFARA 2006). There is evidence that recombination arrest between the rnamrnalian sex chromosomes evolved in successive stages. as the above model predicts. This is deduced front the observation of X chromosome “evolutionary strata". The strata are genomic regions with different divergence between homologous X and Y gene pairs. with the least diverged region (indicating the most recent reoombinatitm arrest) located closest to the PARI on the genetic map of the X. and the most diverged (in which recombination between the X and Y ceased longest ago) furthest from the PAR] (LAME and PAGE 1999a: SA. NDSTEITl' and Tl. !Cll£lt 200-8). The gene order of mammalian X chromosomes is highly conserved. whereas the Y chromosomes are very reamtngcd. which suggests that inversions have oocun-ed on the Y during these chromosomes‘ history. estimated to be about 300 MY (LAHN and PAGE I999:r). The divergence values are not discontinuous enough to define boundaries definitively indicating inversions. so loss of crossing-over may have involved reanangentents and/ or gradual recombination arrest (Ross 2! rd. 2005: lwAst: el al. 2003: MAR/ us and G: L'l1ER 2003: SKALETSKY el al. 2003). Mammalian X-Y systems are ancient. and thus not suited to gaining ll detailed understanding of the early stages of this evolution. or for testing the sexual antagonism hypothesis. The recent evolutionary history of flowering plants (LEEBEV5-MACK cl al. 2005). and the scattered distribution of dioecy (individuals with separate sexes) among them. suggest recent evolution of dioecy in angiosperrns. sometimes even within plant genera such as the
  7. 7. Silrne lruifnlia male function genes. p, 7 gents Silent (DESFIZUX rt ul. 1996). yet some dioccious plants have X-Y . ~cx chromosome systems (WESIERGAARD 1958; CllAl(Lt5Vt'0R'l‘ll 2002: Amswoktn t‘! at. I998). The early events in X-Y evolution can thus be studied in plants (reviewed in VYSKUI‘ and HOBZA 2004, NFLIRUITLJ er al. 200]). Dioecinus species of the genus Silent: have started to be studied in detail. 11teir sex chromosornes are estimated to have evolved recently. only about If) - 20 MY ago (NICOLAS el al. 2{X)5; BERGERD er al. 2007). The available evidence suggests that these Y chromosomes are not highly degenerated: the DNA is not hypcmtcthylated (SIROKY el al. I998). nor is there histone H4 hypouectylution compared with the X (VYSKOT et aL I999). The non-recombining Y region includes most of the length of the Y. which is longer than the X. and a PAR region is on the q arm (I. £N(jERUVA cl al. 2003). Partial deletions of the S. lrmfolm Y chromosome have revealed three regions controlling different sex functions. Ono region suppresses female an organ development (deletion straim have hermaphrodite flowers; Wusmxoutno I946; LARDUN 4'! al. I999). while deletions of two other regions lead to anther arrest (deletion strains bear asexual llowets in which male stt'uctt. Irc. ~ closely resemble those in females; DONNISON r’! :11. I996: FARBOS rt al. I999) or late male-sterility (WE-‘.311-‘RGAARt) I946). mspectively. showing that these regions atny genes involved in male sex organ development and male fertility. rc<peetively. The generally accepted cytogenetic model (Figure! ) is that the regions corresponding to helmaphroditie (female suppressing) and asexual (early stamen promoting) phenotypes contain the two xex detennination functions which are responsible for the sexual dimorphism in this species. Figure I hen: More detailed dissection and localization of different functional regions of the Y chromosome is now possible with the help of molecular markers. Sex-linked markers in S. Iaufoliu (Supplementary Table S1; Figure l) have been used to estimate both the
  8. 8. Silene lau'j’olt'a male function genes. p. 8 recombination map of the X and relate this to estimated X-Y sequence divergence (NICOLAS er «I. 2005; FtL»'lV0V 2005a: BERGERO er al. 2007) and to compare the X and Y gene orders using deletion maps of the Y (LEBEL-HAIIDENACK el al. 2002; ZLLTVOVA at al. 2(ll5a). and to understand the origins of the sex ehmrnnmme pair and whether translocatinm from other chromosomes have been involved (Frmmv 2005»: MARKOVA er at. 2006). These results suggest that, similarly to the mammalian sex chromosomes, restriction of recombination between the S. larrjfolia X and Y evolved gradually (Nl(Y)LA§ er al. ZII5: HLATOV 20053; BERGERO er al. 2007). ‘They further suggest that rt large inversion inferred on the Y chromosome may have caused the observed rearranged order of genes. but probably did not cause X«Y recombination arrest tZuJvovA :1 ul. 20052). since the genes within the inverted region include pairs with a wide range of X-Y silent sire divergence values. Here. we study a set of I6 S. Iatrfalia Y chromosome deletions described previously (LARDON 1'! al. I999: FARBOS er al. I999). plus I2 new deletions. For each deletion which affects male functions. we describe the anther and pollen phenotypic defects in detail. in order to estimate the number of genes carried on the Y ehromosorne whose loss abolishes male functions. These enuld he genes in two categories (i) genes with copies on the X whose X alleles cannot replace the Y gene functions (suggesting that the Y allele has become more male-pmmoling, or else that the X carries an allele that has weakened in male function: among the latter will he the gene in which it mutation caused the appearance of females in the species). and iii) genes with no X copies. such as the Y-linked genes in Drosophilxt (CARVALIIO 2(X)2). We also tested each mutant for the presaertce or absence of molecular markers. to find out which of them are due to deletion of Y chromosomal regions. The mapping work reveals no inconsistencies with previous maps of the Y, and largely oortfrnns previous conclusions. This allows us to base our conclusions on the best ordering of the deletions so far possible. as
  9. 9. Silcne lau'j’olia male function genes. p. 9 well as to demonstrate Y linkage of the new mutants studied. Finally. we examined X-Y chromosome meiotic pairing in all the mutants and identify three Y deletion mutants exhibiting altered X-Y pairing during meiosis. MATERIALS AND METHODS Rationale and methodology of the screen The results in this paper are based on screening deletion mutants obtained by irradiating pollen grains, pollinating wild type females with irradiated pollen. and screening for abnormalities in the M1 generation. Reoessive mutations on the autosornes or the X chromosome should be masked by their wild-type alleles in the matemal genome. whereas dominant or dosage—sensitive mutations in autosomal and X-linked genes will be detected in the screen. Plants with deletions of Y-linked genes which do not have functionrrl homologues within the genome (type (i) in the list above) will also show phenotypic defects. as will plants with rnutatiom in genes of type (ii) listed above. but recessive mutations in Y-linked genes with functional homologues within the genome will show no phenotypie defects. Y chromosome deletions can be distinguished from autosomal and X-linked mutations by using Y—lirtlted markers. Thus. although a large number of genes may exist on the S. Ialifnlia Y chromosome. our screen detects rt specific category of interest, those with no functiortal homologue: within the genome. One mutant (1115. see below) arose spontaneously in a family grown from it cross between plants from two laboratory strains provided by Dr. M. Ruddat (University of Chicago). All the other mutants were generrted by gamma-irrrr. liation of pollen from a single male plant. which was used to pollinate a single female parent (LARDON er al. I999). The mutant plants were vegetatively propagated. and flower buds of several replicate individuals were analyzed for each deletion.
  10. 10. Silene Iatifolia male function genes. p, I0 Y-specific markers were used to confirm the presence of Y deletions. and to localize them on the previously published 5. Iurrfufia Y deletion map (ZUUVOVA er al. 2005a. Hotru rt at 2006). Details of the markets are shown in Supplementary Table SI. which includes references to tlte publication-i in which the conditions for the PCR amplifications are described. All the markers that we genotyped in the mutants are completely Y-linked. Cytological and histological analysis Mitotic and meiotic preparatiom were pcrfomted as described in LARDON el al. (I999). Flower bud fixation. pantplast embedding. histological preparations. and staining were perforrned as described previously tzwvova (1 al. 2005b). Preparations were observed using an Olympus Provis AX70 microscope. and pictures were captured using Isis software (Metasystems. Sandhamen). RESULTS Generation of Y deletion mutants with sex phenotypes We expect our screen to recover mainly dominant or partially dominant mutations due to deletions or mutations of Y-linked genes that have no functional homologue: elsewhere in the genome (see Methods), i. e. either null mutations or deletions (which are expected to behave as nulls). The screen of 2.262 MI plants identified a total of 36 plants with sex function mutations. 16 were described previously (I4 hennaphrnditic bu mutants. one of them uutosomul. and two asexual asx mutants: LILRDON er al. I999: FARBOS er al. I999). Here we describe for the first time I2 suunen mutants (out of the remaining 210 mutants with sumen defects) for which we also demonstrate Y linkage below. No other morphological defects in floral traits. other than one lloml honteotic and meristic mutation (SClI'lT er al. I999) or occasional albino. dwarf. or narrow-leaved plantlets were observed (L-RDt)N er al. I999).
  11. 11. Silt’! !! Iau'j'olt'a male function genes. p, I I Thus. male defects are found disproportionately. compared with other phenotypes. This is consistent with these mutations being due to Y chromosome partial deletions. Here. we characterize the male defects of the 12 new deletions and add more details to the descriptions of those previously studied. Our new histological analyses disclosed defects in a broad range of anther developmental They include mutants with alterations in the early formation of the anthers. referred to as anlher develnpmenl mutations (ad! — 7). two of which — denoted here by acid and ad7— were preliminarily described as “mi. tpan'etn“ mutant: (mspl and 2 of Hl. lNISDAl]. S er al. I997). and in micrnetpure matumtion (pollen fertility mutations. denoted by pfl — 4). The b. l‘. ' mutants are almost all male and female fertile (LARDUN er al. I999). but all mutants in the other categories above (an. ad and 11]) are male sterile. The mutants have maintained their distinctive phenotypes when repeatedly propagated through cuttings (at least I0 vegetative clones per mutant in the last I0 years. for all mutant subgroups in the an. ad and pf classes). Metotk behavior at mutants with stamen and pollen deieets ln most mutants. meiotic X-Y pairing was unchanged (Fig. 2A). hut abnormalities were seen in three independent lines. Two mutants. M2 and ad}. showed variable rates of interstitial X- Y pairing. suggesting an inversion including the PAR region (Fig. 2B). while ad7 exhibited a high frequency of X—Y pairing (84%). including extensive pairing in X»Y regions that never normally pair (Fig. 2C). Figure 2 here Deletion of Y chromosome markers The mutations are inferred to be due to partial deletions of the Y chromosome (see below). and. in some mutmts. deletions are cytologically visible (Table I). The largest deletion
  12. 12. Silt’! !! laufolia male function genes. p, I2 causing loss of male functions. (ml. lacks roughly 40% of the p arm of the Y (Table l: FARBOS er ul. 1999). Most of the p arm (the arm that does not pair with the X) is covered by deletions detected in our screen. Deletions leading to herrnttphmdite mutants have previously been estimated to extend to as much as 50% of the wild type Y chmmnsome p arm (the largest is the bu! mutant reported by LARDON :1 al. 1999). Since these plants are largely male-fertile. gene. -t controlling stamen development and pollen fertility functions cannot be located in the part of the Y covered by these deletions. Table I here We tested each mutant strain for the presence of 13 previously described Y-linked markets. six genie and seven anonymous (Sup-plernenurry Table SI). The two an mutants. and the I2 pi and ad mutants. (6 of which have no cytologically detectable deletions). were found to lack at least one of these markers. conlinning that these involve deletions of pans of the Y chromosome. Figure 3 outlines the deletions found in the mutants. Figure 3 here Histological characterization of the mutants The histological results for an. ad and pf mutants are stnnmarized in Table 2. and a more detailed descriptirm of mutants lacking one or more markets is given below and in the Supplementztry materials (Figures SI and S2). The histological analyses of anthers define a variety of clear-cut male sex defects. The first two major categories. rmexuals (asx) and anther developmental mutants (ad). have early anther developmental defects. while the third one includes pollen fertility mutants (pf). Vlfttlt detailed phenotypic data, these categories subdivide into a total of nine groups.
  13. 13. Silme Iatifolia male function genes. p. I3 Table 2 here The asexual class. Anthem of both a. rxI and a. rx2 mutants are similarly nnested before differentiation of the parietal layers and the mutants are that completely sterile. These flowers have suppressed gynoecia, as in wild—type male flowers. and rudimentary anthers. similar to those in developing flowers of wild-type female (see I-‘Mutos er al. 1999; Fig. 4A shows the wild-type female anther rudiments. and Fig. 4B shows asxl). The anther developmental class. The seven mutants in this class tad! — ad7) have anthers arrested at early stages during the fomtation of parietal and/ or sporogenous layers and are therefore categorized as anther developmental mutants. The ad class includes deletions affecting different stamen whorls and distinct stamen parts. and different anther tissues are affected. and the timing of the defects also differs (Table 2). ‘mesa mutants are again completely sterile either because they do not fonn pollen grains (m1'2. ad-I. ado and M7) or because the few pollen grains produced an: sterile (adl. ad} and adj). These mutants can be further sub-categorized into four distinct and stable phenotypic groups with specific defects. as follows. (i) The ad! rnutnnl has two types of defects. The inner. anti-pelrtlotts sub—whorl of anthers reaches only the four-lobed stage ( i. e. the parietal layers are not yet fonnetl. similarly to the an mutants). such anthers accumulating deposits around the connective region (Figs. 4C and SZL). The outer. anti-sepalutts anther sub-whorl forms ull unther layers found in wild-type plants (i. e. epidemiis. emlothecium, middle layers. and uipetum). but the pollen grains are abnormally small and non viable (Figs. 4Q mad S2K; for the wild-type. see Fig, 4P). (ii) In the ado mutant. developmental arrest occurs at the stage corresponding to the formation of parietal layers in the wild-type (for the wild-type. see the Fig. 4D). Although only the
  14. 14. Silene lau'j'oli'a male function genes. p, I4 primary parietal layer is formed. the microspore mother cells can undergo meiosis (Figs. 4E and SIE). The M2 phenotype is very similar (Fig. SID). (iii) The ad! mutant forms asymmetrically developed anthers — the adsutial locules are well developed (i. c. all anther layers. and micrnspores, form). but the formation of ahaxial locules is highly reduced (Figs. 43? and SIG). The 11:15 mutant displayx a similar asymmetry and phenotype (Fig. Sll-ll. (iv) In the M7 mutant. the secondary parietal layer often appears to differentiate directly. without formation of all anther layers (Figs. 4H and SlKl. The stage of developmental arrest corresponds to the formation of the secondary parietal layer in the wild-type (Fig. 40). Mutant ad! has a similar phenotype. except that anthers are highly variable while degenerating (Fig. Sll). The pollen fertflty class. In the "late" mutants with impaired pollen fertility (pf! - 1115). all anther parts are fomted. but the miemspores either degenerate after meiosis. or else the pollen grains are sterile. These five independent pollen fertility mutants belong to four distinct phenotypic groups. each mutant having a defined tapetal defect. as follows (see also Table 2). (ll Mutant pjl forms all anther layers (similarly to the wild-type‘. Fig. 4|). but the tapetum does not differentiate (Figs. 41 and S23). (ii) Mutant [112 forms all layers properly. like the wilr: Hype (for the wild—type. see the Fig. 4l(). but the tapctum is released from the degenerating middle layer (Figs. 4L and S2D). (iiil Unlike the wild-type (Fig. 4M). mutant pf3 has microspores irregular in shape (Figs. 4N and S2!-‘l. Mutant pf5 has similar defect; (Fig. S26). (iv) In mutant pf4. all parts of the a. nther are properly fumed. but later on the middle layer and tapetum do not degenerate (Figs. 40 and S21).
  15. 15. Slime Iati_[olia male function genes. p. I5 Location of Y regions with stttmen lnrllspensahle functions To determine the number of male indispensable genes responsible for the mutant phenotypes. we ascertained their locations by arranging the Y-chmmosome deletions of each phenotypic group according to the presence or absence of molecular markers. The deletion results show that male indispensable genes are located in only two or three Y regions. On the [2 am, there are one or two regions containing the loci causing the an and ad mutations. i. c. genes with mainly early starnen functions. All the pf loci (affecting late rrutle functions) locate to one region on the 1] arm. The overlapping series of deletions at the distal end of the Y diromosome p arm (Figure 3) produce hermaphmditic (brx) mutants. "flies: deletioma (up to 50% of the chromosome arm) indicate that the gynoecium suppressing factor. or female suppression sex deterrnination gene of males (often denoted by GSF or by 344'). is located at this end of the chromosome (LARDON el al. 1999: also see Farusos er al. 1999), consistent with previous results (WFSIIERGMRD I958). The loci that. according to the wrrent deletion map (Flgln? 3). are closest to the GS! ’ region. correspond to mutants M5. 6 and 7 (which are oo-deleted with the SeDO5 marker). bttt the physical distance between the two classes of genes is unknown. ad-type defects are never seen in but mutants. The size range of the brx deletions suggests that the distance between GSF and the closest ad loci must be larger than 50% of the p am. As explained above. the cytogenctic results indicate that the GSP-containing region of the Y does not contain genes controlling stamen development or pollen fertility funetiorts. Consistent with the cytology. several markers that are never deleted in ltennaphrodites, have been shown to be co-deleted in stamen function mutants (figure 3). SCQI4 lZllANG er al. I998) has thus been inferred to be close to a stamen promoting factor or factors (ZUJVOVA (I al. 2005a) and SIY4 (Armassov el al. 200l) to a male fertility factor or factors (Moon: er al. 2003). Interestingly. plants with at range of male defects lack the SCQI4 marker. including
  16. 16. Silrne lcut'j’nlia male function genes. p. I6 all the asexual and anther-developmental mutants (rm and ad! M7). and also one of the pollen fertility mutants (pf-t. see Figure 3 and further details below). In contrast. the deletions in the four mutants with only pollen fertility defects. pf]. (:12. M3 and (2)3. defining a locus or loci involved in late anther functions. lack the Sll’-I gene, which is currently mapped on the q- arm. At all these deletions are cytologically small (Table I. Figure 3). these loci must be located close to SIY-I. in a dilTerent region of the Y chromosorne from the SCQI4 marker. The tux and ad mutants thus probably have overlapping deletions around the . S'cQI-I marker. which may extend to tlilferent distances on either side (the physical sizes of the regions between S¢‘QI-I and the adjacent markers on either side are unknown. and could be large). 'l'he («I5 and add mutants apparently also have a second. more distal. deleted region. partially shared with the (M7 mutant (defined by the three consecutive markers. MS-I. SrD05 and SD’). see Figure 3). Mutant ad5 lacks the lirst two markets. and ud7 lacks the second two. while ad6 lacks only the marker believed to be in the middle, Sl‘D05. Thus the abnormal X-Y pairing. which is seen only in (1417. could be due to Ims ofa gene (or sequences) located in this distal region. since the deletion in this mutant appears to be more extensive on the side where SIY3 was mapped by Zl, L?'0VA at al. (2005aL Altematively. this gene or sequences could be located near the St‘Ql-I region. since ud7 may have a deletion that extends beyond the region deleted in the other ad mutants. but not its far its SIY4 (since tlte pf mutants have normal chromosome pairing). The mutants osxl and a: x2. in which. as just explained. SCQI4 is absent. have large deletions of the Y chromosome p arm (Table 1). These rnutzutls lack the sturnempromoting sex determining function. often denoted SPF (FARBOS rr Ill. 1999). Since a. rx2 has a cytologically smaller deletion than tux}, the stumen promoting sex-deterrnining gene must lie within the region corresponding to the nu) deletion (249? of the p arm of the Y). and the distance between ScQl-I and the SPF locus (and it gene or genes essential for early stztmen development. defined by the ad mutants) must thus he < 24!? of the p amt.
  17. 17. Silt’! !! lau'j'olia male function genes. p, I7 The probability of all the deletions with the ad phenotypes occurring in one region of the Y, and all the pi deletions in another single region. is very low, if each of the eight ad and pf groups (Table 2) is deleted for a different gene. assuming that the genes are randomly distributed on the Y chmmosomc. We discuss below possible explanations for the finding that then: are apparently two dizninct gene regions on the Y chromosome with indispensable roles in anther formation, one containing the as): and ad loci on the [1 arm (early stnmen developmental factors). and one bearing the pfloci on the q nnn (pollen fertility factors). DISCUSSION Our screen identifiul nine different phenotypic groups of Y-linked mutants with a broad range of stamen development and pollen fertility traits (Table 2). in addition to the sex determination functions corresponding to the an and bu mutations previomly described. Defects in late stamen functions. notably tapetum malfunction. like those in our pf mutants. have been reported repeatedly in various plant species (SANDERS et ul. I999; Vlzttt er al. I994; GORMAN and MCCORMICK 1997). The an and ad mutants are, however. unusual. In Arabidopsir Ihaliana, only a few mutants have been identified with defects in the specification. organization or development of anther cell types. similar to those in our ad chm (e. g. (M2 and M4; see SANDERS er al. 1999; Vault et al. 1994). Mutants with such defects were also not found in large-scale screens for fertility defects using EMS or irradiation mutagenesis in other model species such as maize and tomato (NEuH= t=. k er al. 1997: Germ»: and Mccouucx 1997). This suggests that the S. latrjfialia Y chromosome carries an unusual kind of locus with early stamen functions (or an unexpectedly large number of such loci). in addition to a locus that generates the more common type of mutations affecting late stamen functions.
  18. 18. Silme Iatifolia male function genes. p. I8 Types of male-hrllspensable genes ltlentlfled Since several types of mutations appeared more than once. the Y has probably been saturated with respect to mutations affecting indispensable male functions. ie. all regions whose deletion can affect such functions have been identified. Otrr mapping of atx. ad and pf classes shows that the corresponding deletions span fairly large Y chromosome regions (Table 2). The observed an - ad phenotypes. produced by overlapping deletions, can thus be explained in the following ways (similar reasoning can be applied to the pf deletions). (I) One pomihility is that the different phenotypes observed with deletions in it single region resulted from deletion of different loci. i. e. indicating that several different genes with male indispensable and closely related functiom lie in this Y region. (2) Altematively. the region oontains rt major gene involved in early stzrmen development. plus other (modifier) genes that interact with this gene. so that the phenotype is different in each distinct deletion. We cannot distinguish between these two possibilities. A variant of (2) above is that effects such as position effect variegntion (PEV) could influence the expression of non-deleted genes located near the breakpoints. The fact that independent mutants within a group te. g. the ad} and «M5 mutants) were always phenotypically identical or very similar, suggests that PEV is unlikely. since it generally leads to variable phenotypes with the same rearrangement (WEILER AND Wntrxrmo, I991! and refs therein). Male speelalntlon of the Y The screen itlerrtified almost exclusively male function genes. and the mutant phenotypes are dominant. or at least semi—dominant. Dominant mutatiom are generally much less frequent than recessive ones ( for an estirnate in S. Ialrfolia. see LARDON el ul. I999: also see ORR 1991). so that it is not expected that one will find many such mutations in it screen.
  19. 19. Silcne latij’olt'a male function genes. p. I9 For male reproductive functions. however. the theory outlined in the Introduction predicts that Y cluutnosomes will be enriched for such genes. either due to (i) evolution of Y-linked alleles which actively increase male functions relative to their X—lin| :ed homologues (so that loss of the Y copy impairs male function). or (ii) because of transposition of genes onto the Y but not to the X: in addition. loss of the SPF should lead to complete sterility. since the X chmmosorne has a nonvfunctional allele at this locus (defined as the locus that is mutated in females and causes their male sterility). Y chromosome genes of either type (i) or (ii) might have deleterious effects in females. and fit into the category of sexually antagonistic genes proposed by RICE (I987) that are expected to accumulate on the Y once diuecy evolves. In S. lanfulia. the SIAP3 Y gene is already known to be an example of recruitment to the Y of an autosomal gene. whose function has subsequently become more male specialized (MATSUNAGA er nl. 2003). This gene appears to map in the a. t'. t-ad region (figure 3). Although it is not possible to tell which of the two categories of changes mentioned above has been involved in cases other than this. the results described above suggest that the S. lat(fm't'a Y chromosome has started to undergo the predicted changes and eanies several genes pmmnting male reproductive functions that cannot be replaced by X- linltetl alleles or autosomal genes. i. e. has evolved some male specialization. Further aecentuation of tlte process may also result from Y-associated gene loss through degeneration and subsequent deletion of other genes (such as the MROSJ gene: reviewed in CHARt. F.swott'rtt and CHARLESWORTI-I 2000). but there is so far little evidence for this in S. lattfnlia. Early and late suunen functions ddlne distinct Y regions According to the hypothesis proposed by CHARLESWORTH and CttAttuaswoR'r1t ( I978). male-benefit genes should sum to accumulate on the pruto-Y chromosome while it still recombines with the proto-X. forming clusters around the initial sex determining genes. Once
  20. 20. Silene latij’olia male function genes. p. 20 a non-recombining region has evolved. male-benefit genes will continue to evolve throughout the Y-specific region [RICE I987). Each of the phenotypic categories of Y—linked mutants described above (bsx. asx-ad and pf) is associated with deletions of specific parts of the Y eltrnmosome, and the three major categories of mutants are each found only in deletions of a single Y region. These results thus suggest only two distinct regions with Y-specific genes having roles in anther formation. One region. located on the Yp arm. contains the gene(s) responsible for all nine early stamen developmerrtul mutants. including the an mutants and the four ad groups defrnal in Table 2 (all nine of these mutants are deleted {or the Sr: Ql4 marker. see Figure 3). In contrast. four of the five late stttmen mutants (pf) have deletions in it dilfcrent region. on the q arm. defined by loss of the SIY4 marker. This distinct puttem would be highly unlikely if these mutants represent I4 genes scattered throughout the Y cluomosome. The female suppression region (containing the GSF gene defined by the bu mutants) is large (about httlfof the distal Y chromosome p nrrn) but contains no genes indispensable for stamen functions. A stztmen promoting region is defined by the rut mutants. This region. including the genes indispensable for early stamen functions which are deleted in all seven ad mutants and one of the five pf mutants. defines the proximal ~ 20% of the p arm. It may include the gene that is mutated or absent in females that causes their male-deficiency. which may correspond to the locus defined by our asx mutations and thus represent the Y chmmsomal SPF gene. If the an-ad region contains several different genes with male- indispensable functions (see above). any of them could play the rule of ti male sex determination function. and could thus he the SPF gene. Frnzrlly. very lute functions involved in pollen fertility (defined by the pf mutants) are located distant from the sex determination regions in the current S. lalrfoha Y. close to the breakpoint of the large Y inversion inferred to lie between SIYI and SI}’4 (Figure I: see also below). The inversion must have occurred recently. as genes with high and low X-Y sequence
  21. 21. Silt’! !! lau'j'olt'a male function genes. p, 21 divergence (NICOLAS er al. 2005: FILATOV 2005b) are found in adjacent locations in the Y map (ZLIJVOVA el al. 2005a). If the recent anoestml state was the same gene order as the current X chromosome. the pfgene(s) must thus have been located either near the SIYI gene, i. e. close to the PAR on the q arm. or else at the end of the arm distant from the PAR (i. e. near the GSF locus on the p arm). On the second hypothesis. the inversion (Figures I and 3) may be reinforcing reduced X-Y recombination. either directly through the well-known effect of inversions in causing lower survival of reeornhinant ytrncoes or zygotes due to chromosome breaks. or as a secondary consequence of ullowing sequence divergence of this region of the Y. Melotlc X-Y palrlng Three of our rrtutmtts have altered X-Y pairing in meiosis. Among them. the (1417 deletion clearly indicates that the Y chromosome carries elements or functions essential for X-Y pairing most. 5. lanfolia mutants with pairing along the scx chromosomes may have been observed by Wt= .s1'FJtcAAttt) (I946; type Y3. with the ditlerential. or p am only about one fourth as long as the pairing am). but he interpreted his cytological observations in terms of a translocation of a part of the X chromosome to the Y. with loss of almost all of the Yp arm. This interpretation cannot explain the 41:17 deletion. because this mutant's Y chromosome is only slightly smaller than the wild type Y (Table I) and still most of the Y-specific markers we tested. including market: from mast Y regions (Figure 3 and Table S2). The cause of the lack of pairing throughout most of the X and Y chromosomes is unknown. A re- inversion in the mutant of the putative inverted region ol’ the Y chromosome (Figure 1: Zwvovn er at. 20053) seems implausible. If this is correct. the ud7 mutation indicates [hill the Y carries gent. -ts) or sequences restricting X-Y pairing. It is not possible to test whether pairing in the «M7 deletion ullects X-Y recombination. because the mutant is rrutle-sterile.
  22. 22. Silt’! !! lau'j'olt'a male function genes. p, 22 Restoration of recombination seem unlikely. given the large sequence divergence that is likely in this region of the Y. The region deleted when the pairing function is mutated is not close to the pseudo autosomal region (which might suggest that it was involved in loss of recombination in the most recently evolved ‘sttatum' of the sex ehrommornes). lnterestingly. however. it is close to the region carrying the SIY3 gene (Figure I), a gene pair with the highest level of X-Y sequence divergence yet observed (NK‘. nLMS et al. 2005; BERGERO er al. 2007). As explained above, this region also contains a male promoting gene. which could be one of the primary sex determination loci. In this region, the S. Ianfolia X and Y chromosomes are as highly diverged as homnelogous chromosome pairs in many allopolyploid plant species. This lack of close homology may not necessarily prevent chromosome pairing. A eltnasical example of homeologous chromosome pairing occurs in wheat. and it is suppressed by an allelic variant of the Phi locus (RILEY and Crumb»: l958:MArt1'rNr~1-Pzruz el :11. 200i: Crltlfi-T1115 er al. 2006). It is possible that the S. lalifofla genome has evolved it similar mechanism to restrict sex chrummorne pairing. Whatever the mechanism. our results are consistent with restricted X-Y recombination in S. lalrfuhu having evolved in steps. as suggested by previous results on differences in sequence divergence of X-Y gene pairs spanning most X-Y regions (NICOLAS (I at. 2(l)5'. FILATDV 2t'll5a. BERGERO er al. 2007). The meiotic behavior of the M7 mutant supports this. by indicating that pairing can be stopped through sex chromosome pair-specific processes. In S. larrfoliu. more genes (for estimating X-Y divergence. which can he used to infer when recombination ceased). and FISH experiments with BAC clones. will in future allow better inferences of the extent of X-Y rearrangements. including detecting other, smaller inversions that may have occurred at different times in the evolution of the Y chmrnosorne (some of which might have been involved in suppressing its recombination with the X).
  23. 23. Silrne Iaufnlia male function genes. pr 23 Acknowledgments This research was supported by the Grant Agency of the Czech Republic (20-"I/ D5/P505 to JZ, 52l/ fl5I‘2076 tn Bl, 521/06/0056 In BV). Institutional Research Plan (AV07.50()-$0507! and through the bilateral Fn: nch—Bulg: u'ian program Rila063 l6YC. DC’-4 wart was funded by the Natuml Envimnment Research Council of the UK.
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  31. 31. Silene laufolia male function genes. p. 31 VICOSO. B. . and B. CHARLESWORTH. 3006 Evolution on the X chromosome: unusual patterns and processes. NnL Rev. Gena 7: 6454353. V| zlR. I. Y. . M. L. ANDERSON, 7. A. WILSON and B. J. MLILLIGAN. 1994 Isolation of deficiencies in the Arabidnpulr genome by {gamma}-irradiation of pollen. Genetics I37: llll-Ill‘). Vvsxm. B. . J. Suzoxv. R. HLADILOVA. N. D. BELYAEV and B. M. ‘manual. I999 Euchmtnutic domains in plant chromosomes as revealed by H4 liistune ucetylntiun and early DNA replication. Genome 42: 343-350. Vvsxo'r. B. . and K H0374. 2004 Gender in plants: sex chromosomes are emerging from the fog. Trends GeneL 20: 432-438. WEILE-LR. K. S.. and B. T. WAKIMOTO. I998 Chromosome rearrangements induce both variegated and reduced. uniform expression of helerochrommic genes in a developmental- specific manner. Genetics I49: I45I-I464. WESTERGAARD. M. . I946 Aberrant Y chromosomes and sex expression in Melundriunv album. Hereditas 32: 419-443. WESTERGAARD. M. . I958 11): mechanism of sex delerrninnlion in dioecious flowering plums. Adv. Genet. 9: 217-28l.
  32. 32. Silene laufolia male function genes. p. 32 WINGE. 0.. I927 The location of eighteen genes in Lebtltm retirulatus. J. Genet. 18: I43. HIANG. Y. H. . V. S. DI STILIO, F. REHMAN. A. AVERY. D. MULCAHY el UL, I993 Y chromosome specific markers and the evolution of dioecy in the genus Silene. Genome 4|: I41-I47. ZLUVOVA, J. . B. Jmousax. I. NEGRIZTIU and B. Vvsxm. 2005a Coniparisan of the X and Y chromosome organization in Silene laltfnlia. Genetics I70: I431-I434. Zwvovx. J. . M. LENGEROVA. M. MARKOVA. R. HOBZA. M. NICTLAS el al. . 2005b The inlet- specific hybrid Silent Iarrfalia x S. viuma reveals curly events of sex chromosome evolution. Evol. Dev. 7: 327-336.
  33. 33. Tnhles TAILS l. Silene Iaufolia male function genes. p. 33 Cytological characterization of ma| e~defective mutants of S. Iatifolia. The 2"‘ column of the table shows the sizes of the Y deletiom measured in mitotic cell preparations. as a percentage of the entire Y chromosome. An extcnr-ive meiotic analysis was performed to estimate bivalent fonnation m during diakinesis and metaphase I (columns 3 and 4). other meiotic alicmlions (column 5) and X-Y pairing behavior (column 6). Sin: nl‘ Nlciuix Mutant narrm debtiorl Other chimes (‘ll Dialsinesin Melzphane I X-Y pairing ad] 9 I2 I2 wild-type ml. ’ I2 I2 I2 no or illevslilial (arm I09! abnormal rut? ml I2 I2 hnerxtilhl Illfilal nyegnlicn in Incatsurahle meiosis II ndl I3 I2 I2 wild-type mt! ml I2 I’ ‘Id - w -I measurable ‘ we pf! -4 I2 I2 wild-type Du p}? not studied not studied Ivilcl-type measurable pf! M‘ II I’ ‘M I - w - nnarurabl: ‘ ype Mitotic P“ not I 2 and I3 di _ _ l_ _M n on 0 in -l meutnble (l0*i) 5', x we one bnnlent III! pfS I4 wild-type mensunhle adfi (Irupl I 8 I2 I2 wild-type ml? rraupzl '2 '2 cm-mive X-Y pairing (84%) an! 2!! No rneiorskx not Iemhk “:2 I2 No meiosis not Icuultle (a) see LARDUN el ul. (I999): number ofhivalents are known to vary from 2n = I2 to 2n = I4 in S. lanfuliu genotypes with no consequences on sexual phenotype.
  34. 34. TABLE 2. Chttline description of the phenotypes ofthe deletion mutants. and suggested functiontsl of the genes involved. The left-hand columns list the three major male-defective phenotypic clmses and show the total numbers of deletions found in each of them. The 3'“ column describes the general phenotype oleach group (numbered with roman numerals). Mtttttm class and Y deletion sizes uexul Interstitial, tip to 40% of Number of deletion rltulanus 2 Mutant group ntunes. Phenotypic defects possible gene functions involved in each sub-group (numbered with roman numerals) uni. tux) / tntlter arrest before tlifferentiation of the parietal layers. as in wild-type females. The Splecfifying primary mrletal rtwi tnutants have an identical phenotype. «:2 ate. Figures 4B. SlB Exintal Yp anther 7 ad! — ad7 Antbers arrested early during lonnutiort of parietal and spotogenoto layers. development Conoletel y sterile. Interstitial. up (I) ad] Inner. antipetalous. arttlier sulrvaltorl is arrested at the four-lobed stage. deposits 4C. 4Q. to 26% of Stanen sub-whorl aocumulate around the connective region. Outer. anthsepalous anthers form s2|(_ proximal Yp coordination abnormally small and invinble pollen grains. 53L (ii) adi’. add Anther arrest at the stage of parietal layer forrnation in the wildtype. but tnierospore 4E, S l E, Maintaining parietal layer mother cells can undergo meiosis. The two mutants have very similar tiienotype. 5 | D (iii) M1. M5 Artyrnmetrically developed anthers (abttxial locules form only primary parietal layers). 4F. S IG. Lncule synchronization 11!: two mutants have an identical phenotype. 5 | [1 (iv) ad4. ad7 Secondary parietal layer often dillerentiated directly iruo tapeturn-like cells. without 4H. S I]. Specification of parietal formation of all anther layers. The two mutants have very similar phenotype. 5| K layers ad? is altered in X-Y pairing. polen fertility S pfl — p/ S Micmspores either degenerate after meiosis. or the pollen grains are sterile. Interstitial. up (i) pfl Tapetutn does not difleterttiate. 4]. S28 to 8')! » of Tapetum differentiation proximal or (ii) PF Tapetum is released from the degenerating middle layer. 41.. . SZD meddial Yq Tapetumlmiddle layer signaling (iii); tf3. p/5 Mictospores are irregular in shape. The two mtttanus have very‘ similar phenotype. 4N. SZF. Micnworv st-we SZG (iv) p_/4 Tapetum and middle layertlo not degenerate. 40. S2l Middle layer and tapetnm degeneration function.
  35. 35. I-‘igtre legends FIGURE 1. The S. larrffnlia sex chrommnrnes and the current sex dctcnninatim model according to NEGRUTILI er al. (2(X)l) and CHARLIBSWORTH (2002). Y-chmmosorne deletion mutants (WESTERGAARD 1958: Domnsou er al. I996; LARDON er al. 1999; FARBOS at al. 1999: LEBEL-HARDENACK er al. 2002: 7J. U'0VA :1 al. 20054:) define at least two critical sex- determining regions on the differential arm of the Y chromosome: the regions responsible for suppressing female organ development and for early rstnmen development (see text). while the arm homologous with the X (i. e. containing the PAR) carries it region responsible for late starnen development (plants lacking this region are male-sterile). When compared to the known gene order on the X chromosome. presence of an inversion can be deduced. Molecular markers are listed on the right. those present both on the X and Y chromosomes are in hold. The p and q amts are indicated. FIGURE 1. Meiotic preparntions of wild-type and two Y-deletion mutants showing altered X-Y behavior in meiosis. Wild~type dialtinesis. Note the terminal pairing of X and Y (A). Dittkinesis in M2 mutant in which X and Y do not pair. Note that the distal regiom of the X are not aligned with any of the Y distal regions (B). Ilalrinesis in M7 showing pairing X and Y. One distal region of each chromosome is aligned (C). White nrrowhencks indicate X-Y chromosome pairs. FIGURE 3. Outline of the deletions observed in the mutants analyzed. The markets are shown at the top. ordered according to the Y map (HOBZA er al. 2006). with inverted triangles indicating the sex
  36. 36. Silene latI'j’olt'a male function genes. p. 36 determination functions located on the p arm and a pollen fertility region on the q arm. The pseudo-autosomal region is at the right hand side. The nrutant namm are given on the left hand side. The underlined mutant nnrnes state for mutants with pairing phenotypes. Full litres heside indicate markers present in each deletion mutant. dashes indicate markers that were detennined to be tnissing, and dotted lines indicate that the presence of the marker was not tested in the strain. Deletions are not at scale. Note that in several mutants more than one marker is deleted. I-‘tt: urtr:4. Histological analyses of S. latifnlia wild-type and Y dtmmosotne deletion mutants. An outline of anther development is given on the left hand side. The middle column shows sections of wild—type anthers. and the right—hand column displays sections of anther developmental mutants. Detailed descriptions of the wild-type anther development and Y- chmnrosorne deletion mutants histology are given in the Supplementary Material (Figs. S1 and S2). 1he anther rudiment in female plants stops developing before the formation of the parietal layers (A). The anthers of the Y chromosome deletion mutant aseruu I (curl) strongly resemble female anther rudiments (B). ‘Rte inner sub-whorl anthers of the anther development mutant ad] also do not develop beyond this stage. probably because of the accumulation of deposits around the connective (C). Wild—type male anthers develop the primary parietal layer and the spomgenous tissue (D). In the anther development mutant ado, the primary parietal layer is altered. but the spomgenotts tissue is able to form microspon: mother cells (E). ln the art? mutant. alraxial locule development is arrested prematurely (F). Unlike the wild-type anthers. where no diflerence in the staining ability of the secondary parietal layers is observed (0), the inner secondary parietal layer of the ud7 mutant differentiates into a derrsely-stnirtcd tapetum-like layer (H). In the wild-type anthers. the tapetum becomes densely stained afier the fonrration of all anther layers. and this process is
  37. 37. Silene lau'j’olia male function genes. p. 37 connected with its differentiatiott (I). However. the tapetum is not stained in the pollen fertility mutant pfl (1). During meiosis and tetrad formation. the tapetum starts to degenerate and its cells are released from each other. but remain attached to the other anther layers (K). ln the pf? mutant, the tapetum is released from the anther layers and fttrtm an agglutinate inside the anther (L). The wild type micrmpores. when released from the tetrads, are mund- shaped (M). but in mutant pf3, the mictmpntes are irregular in shape (N). Mutant pj4 farms mund micrnspores. and the endothecial and tapetal cells do not degenerate. as deduced form the fact that the endotheeium is present and that the tapetal cells remain attached to each other (0). When compared to the wild-type (P). both the anther locales and the pollen grairu of the outer sub-whorl of the mutant all are abnormally small (Q). dep. deposits; epid. epidermis: I" par. primary parietal layer: spur. sporogenuus cells: mmc. microspore mother cells: ab. abaxi-at locule: ad. ztdaxial lucule: 2" par. secondary parietal layer: tap, tapetum: mi. microspores; pg: pollen grains. Bars represent I0 tun (A. B. C. E. H. J. O). 25 um (D. G. l. K. L M. N. P. Q). andfiourn (F).
  38. 38. Silt"? Iaflfolia male function genes. p. 38 SUPPLEMENTARY MATERIALS References for Table Sl. ATANASSOV. L, C. IXELICHERE, D. A. HLATOV. D. CHARLESWORTH. I. NEGRUTIU H al. . XXII Analysis and evolution of two functional Y~| inIted loci in l plant sex chromosome system. Mol. Biol. Evol. 18: 2I62-2168. DELICHERE. C. . J. Veusxtms, M. HERNOULI). N. BARBACAR, A. MOURAS er al. . I999 SIYI. the first active gene cloned from a plant Y chromosome. encodes a WD-repeat protein. EMBO J. l8:4l(v9—4I79. DONNISON. I. S. . and S. R. GRANT. I999 Male sex-specific DNA in silent laiifoliu and other dioocious plant species. pp. 73-87 in Sex Dclcmiinatinn in Plants. edited by C. C. AINSWORTH. Bios Scientific. Oxford. FILATOV. D. A. . 2005 Substitution rates in a new Silt’! !! Iaufnlia sex-linked gene. Sl. t.vX/ Y. Mol. Biol. Evol. 22: 402-408. Honu. R. . P. Hnusucova. J. SA! -‘AR. J. Bums. B. JANOUSEK et al. . 2006 MKI7. u sptxifit: marker clooely linked to the gynuecium supprewion region on the Y chromosome in Silent laIifoha.111eor. Appl. Genet. I13: 280-287. MATSLINAGA. S. . E. ISONO. E. KEJNOVSKY. B. VYSKOVI; J. cl al. . 2003 Duplicalivc transfer of a MADS box gene to l plant Y chromosome. Mol. Biol. Evol. 20: 1062-1069. Mootu-: . R. C.. O. Kozvruzva. S. LEBEL-HARDENACK. J. Smoxv. R. HOBZA er al. . 2003 Genetic and functional analysis of DD44. a sex-linked gene from the dioecious plant Silent laufnlia. provides clua: to early events in sex chromosome evolution. Genetics 163: 32 I -334.
  39. 39. Silcne laufolia male function genes. p. 39 NAKAO. S. . S. M. 1sUNAGA. A. SARA]. T. KUROIWA and S. KAWANO. 2002 RAPD isolation of ti Y chnnnosorne specific ORF in a dioeciots plant. Slltllt latifolia. Genome 45: 4I3-420. NKIOIJLS. M. . 0. MARAIS, V. HYKELOVA, B. JANOUSEK, V. LAPORTE er al. . 21305 A gradual rind ongoing process of recombination restriction in the evolutionary history of the sex chromosomes in dioecius plants. Pbos Biol. 3: e4. OBARA. M. , S. MATSIINAGA. S. Nmuo. S. Kxwxno, 2002 A plant Y chromosome-STS marker encoding a degenerate retrotransposon. Genes Genet. Syst. 77: 393-298. ZHANG, Y. H. . V. S. DI Snuo, F. REJIMAN. A. AVERY. D. MULCAHY el al. . 1998 Y chromosome specific markers and the evolution of dioecy in the genus Silent. Genome 4|: t4I-I47.
  40. 40. TABLE SI. 1112 Y-specific markers used for the mapping of Y chromosome deletions Marker Nature of the marker Dednced function Refenence Bglla repetitive sequence No apparent function DONNISON and GRANT I999 DD-MY gene Potential oligomycin sensitivity-conferring protein Mount: er al. 2003 MK I 7 single-copy sequence No apparent function Hoau (1 al. 2006 M84 rermtransposon Degenerated gag protein of a Ty3-gypsy-like retrorransposon OBARA at al. 2002 ScD05 anonymous Unknown ZN-NG er al. 1998 ScI(02 anonymous Unknown ZHANG er al. 1998 SCQI4 anonymous Unknown ZHANG er al. 1998 Scxll anonymous Unknown EIANG at al. 1998 SIAPJY gene MADS box gcnc MATSUNAGA el aL 2(X)3 8188 Y gone Spcmtidinc synthmc FILATOV 2(l)5 SIYI gcnc WD repeal protein DELIGIERE el al. I999 SI Y3 gene Putative C DPK NICOMS cl al. 2005 SIY4 gene F1-nctusc-2,6-bisphosphauuc Armnssnv er al. 200]
  41. 41. Iant/ marker MK17SIssY B 10 DD44Y M84 ScD05 SIAPSY SIY3 ScO14 SIY4 ScK02 ScX11 SIY1 ND ND -0- ND ND 4- + '9 Taluz S2. Results of deletion mapping. + marker ptcscnl: - marker ubacnl: ND not dctcrminod.
  42. 42. Ificutu-: Sl. Histological analyses of S. luufnlia male wild-type and Y chromosome deletion mutants impaired in anther development. Sections through the wild—type S. latifnlia anthers are shown in the left-hand column. The middle and right-hand columns show anthers of the Y chromosome deletion mutants. The anther nrdirncnt in the female plants stops its development before the formation of parietal layers (at the origin of all cell layers in wild-type anthers with the exception of the epidennis). Arehesporial cells are however present (at the origin of sporogenous and parietal layers. they are round-sltztpcd. usually possessing densely stained cell walls. wherein the cell content except of the nucleus is not stained) (A). The anthers of the Y chromosome deletion mutant asarua I (at-. tI) arrested at the stage of the difierentiated epidermis and arehesporiztl cells resemble the female’s anther rudiments (B). The wild-type male anthers develop tapart of the epidermis) the primary parietal layer and the sporogenous tissue. The primary parietal layer cells resemble in size the sporogenous cells. but they are easily recognizable by the fact that they font‘: a continuous cell layer under the epidennis. The sporogenous oells are of irregular shape localized inside the anther locules (C). In the anthers of the anther development mutant a¢f2. parietal layer formation does not go beyond the stage of the primary parietal layer formation. i. e. only epidermis and one subepidennal cell layer are fomtcd. However. the sporogenous tissue fonns microspore mother cells (descendants of the archesporial cells: in wild-type anthers they develop into niicrospuresi. which are normally fonncd when all anther cells layers are developed (cf. the figure Sll for the wild- type anther development). The section is stained by calcolluur white and observed under fluorescertee rnicmscope to better visualize the cell walls. (D). A similar phenotype (i. e. . developmental arrest alter the primary parietal layer formation) was also observed in the mutant ado (previously (HINNISDAELS et al. I997) referred to as mispar-irlu I) (E). The wild- type anther is formed of two ahaxial and two adaxial locules of approximately the same size. The figure shows an anther with all cell layers formed and the microspote mother cells (which
  43. 43. Silcne lau'j’olia male function genes. p. 43 are localized in the inner pans of the locules) undergoing meiosis (F). In mutants ad! (G) and M5 (H). the abaxial locule development is arrested after the primary parietal layer formation (i. e. only one subepidennal layer is formed), but the spomgenous cells are able to continue their development and to fonn micmspore mother cells and undergo meiosis. Both mutants show callosc depositions in the cell walls (visualized by intensely stained cell walls). which immediately precedes the entrance of the micmspnre mother cells in rneiosis. However, the development of the adaxial locules continues as in wild-type plants. During meiosis. all the anther layers (epidennis. enduthecium - the first subepiden-nal layer. middle layers - very thin layers localized below the endothecium. anti tapetum — a densely stained cell layer in contact with the microspure mother cells) are already formed in the wildtype anthers (I). In the mutants M4 (Jr and ud7 (previously referred lo as mi. rparielu2 (HINNISDAELS er al. 1997)) (K). only the epidermis and secondary parietal layers (two layers between the epidermis and micmspore mother cells) are formed at this developmental stage. The inner parietal layer seems to differentiate into the tapetum-like cells because its cells are densely stained. epid. epidemtis; arch, archesporial cells; I “ par, primary parietal layer; spor. sporogenous cells; ab, ahaxial side; ad, adaxial side; endo; endotheeium; ml. middle layer. tap. tapetum; 2" par. secondary parietal layers. Bars represent I0 pm (A. B. D. E. J. K), 25 pm (C. H. I). and 50 um (F. G).
  44. 44. Slime Iarifolia male function genes. p. 44 hcutu-: S2. Histological analyses of S. larlfirlizr male wild-type and Y chromosome deletion mutants impaired in pollen fertility. The sections through the wild—type S. Iatifolia anthers are present in the left column. The middle and right columns represent anthers of the Y chromosome deletion mutants. In the wild—type anthers after the formation of all cell layers. the tqrerum becomes densely stained. This process is connected with tapetum differentiatirrtr (A). However, the rapetum is not stained intensely in the mutant pfl (B). thus it is probably not fully differentiated. During the later wild-type anther development. the Iapetnl cells release from each other (C). In the mutant p12. the tapetal cells remain attached to each other. but release from the other anther layers and form an agglutinare with microspores in the middle of the anther (D). The microspores in the wild—type are round-shaped (E). but they are irregular and densely stained in the mutants pf} (F) and [115 (G). The release of tapetal cells from each other is connected with the middle layer degeneration. At the time of the microspore release fonn tetrads. only epidemtal. cndothecial. and tapctal layers are present in the wild-type anther (H). In the mutant p13. although microspores are present. the middle layer (i. c. the cell layer between the that subepidermal endothecium and intensely stained tapetum) is still present. and the tapetal cells do not release from each other (I). Pollen grains are formed both in the wild-type anthers (J) and in the outer suh-whorl of the mutant adl. However. in the mutant. pollen grains are abnormally small (K). Contrary to the outer sub-whorl. anthers in the inner sub-whorl of the mutant ad! reach only bilohal to tetrnlobal stage. probably because of the accumulation of deposits around the connective (tissue localized on the transverse section approximately in the middle of the anther; it uansnrits nutrients to the anther). The deposits are present in all anthers (L). epi«. L epidemtis: arch. archesporial cells: I ° par. primary parietal layer: spor. sporogenous celk: ab. ahaxial side: ad. adaxial side: endo: endothedum: ml. middle layer; tap. tapetum; 2“ par. secondary parietal layers. Bars represent I0 um (B. H. I). 25 | .tm (J. K. L). and 50 pm (A. C. D. E. F. G).
  45. 45. p-arm q-arm gynoecium suppressing . . . MK 14 sex determining region s; ssy SIX‘ DD44Y SIY3 stamen promoting sex determining region S0074 SIX3 SlssY + DD44X _. _ SIY4 male fertility region SIY1 SIX 1 ScOPA09 ScOPA09 pseudoautosomal region Y chromosome X chromosome
  46. 46. w . . _L___. !'_v'__ __ E ___ i 2 3.3 .5: . IIl. l uI. II'l. l‘. .aI
  47. 47. schema wild type epidermis apldermls ‘ fparletal layer , . 5,}. »‘ V sporoqonous $3.; tissue epidermis '. ' "srparivtal layers sporogenous tissue i <= ‘ Aeepidennls endoilhedum - " middle layer ' ‘ . V _ kgmlcrospooe ' mother cells 1- _ in ~— - K .7 - m , upeturn ' - A'”de9enerateu ' ——* ‘M‘ “ “mlcrosporcs ~ asexual author development pollen lenlllty