Duplicative Transfer of a MADS Box Gene to a Plant Y Chromosome (original) (raw)

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26 February 2003

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Sachihiro Matsunaga, Erika Isono, Eduard Kejnovsky, Boris Vyskot, Jaroslav Dolezel, Shigeyuki Kawano, Deborah Charlesworth, Duplicative Transfer of a MADS Box Gene to a Plant Y Chromosome, Molecular Biology and Evolution, Volume 20, Issue 7, July 2003, Pages 1062–1069, https://doi.org/10.1093/molbev/msg114
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Abstract

Y chromosomes carry genes with functions in male reproduction and often have few other loci. Their evolution and the causes of genetic degeneration are of great interest. In addition to genetic degeneration, the acquisition of autosomal genes may be important in Y chromosome evolution. We here report that the dioecious plant Silene latifolia harbors a complete MADS box gene, SlAP3Y, duplicated onto the Y chromosome. This gene has no X-linked homologs but only an autosomal paralog, SlAP3A, and sequence divergence suggests that the duplication is a quite old event that occurred soon after the evolution of the sex chromosomes. Evolutionary sequence analyses using homologs of closely related species, including hermaphroditic Silene conica and dioecious Silene dioica and Silene diclinis, suggest that both SlAP3A and SlAP3Y genes encode functional proteins. Indeed, quantitative RT-PCR and in situ hybridization analyses showed that SlAP3A is expressed specifically in developing petals, but SlAP3Y is much more strongly expressed in developing stamens. The S. conica homolog, ScAP3A, is expressed in developing petals, suggesting subfunctionalization with evolution of male-specific functions, possibly due to evolutionary change in regulatory elements. Our results suggest that the acquisition of autosomal genes is an important event in the evolution of plant Y chromosomes.

Introduction

Studies of sex chromosomes have concentrated mainly on animal species, since most animals are dioecious. Although most flowering plant species are hermaphroditic, with bisexual flowers that contain both male and female reproductive organs (stamens and pistils), about 6% of angiosperms are dioecious (separate individuals produce staminate and pistillate flowers; see Renner and Ricklefs 1995), and some have heteromorphic sex chromosomes. The Y chromosome of Silene latifolia, in the family Caryophyllaceae, has been extensively analyzed (Matsunaga and Kawano 2001; Negrutiu et al. 2001). There are striking similarities with animal sex chromosomes, as well as some differences. Classical cytogenetic analyses of deletion mutants suggested that the Y chromosome has three functions: (1) suppression of pistil development, (2) initiation of stamen development, and (3) completion of stamen development (Westergaard 1958). Recent studies of Y chromosome deletions induced by γ-rays or X-rays support this general conclusion (Negrutiu et al. 2001; Lebel-Hardenack et al. 2002). Compared with human or fruitfly Y chromosomes, the S. latifolia Y contains little heterochromatin (Matsunaga et al. 1999; Charlesworth and Charlesworth 2000).

It is currently unclear how many X-linked genes have Y chromosome homologs in plants, but two apparently functional Y-linked genes, SlY1 and SlY4, have so far been discovered in S. latifolia (Delichere et al. 1999; Atanassov et al. 2001). SlY1 encodes a WD-repeat protein and is preferentially expressed in the stamens of male plants, whereas SlY4 encodes a fructose-2, 6-bisphosphatase. Both of them have X-linked homologs. Except for the male-determining gene SRY, genes on the human Y chromosome fall into two groups (Lahn and Page 1997). The first group consists of housekeeping genes, which have X-linked homologs. Genes in the second group are expressed exclusively in testes and form gene families on the Y chromosome. The previously reported S. latifolia Y-linked genes are comparable to the human Y-linked housekeeping genes. We here report the discovery of a Y-linked MADS-box gene with no X-linked counterpart.

Extant dioecious species of Silene include a group of close relatives, S. latifolia, Silene diclinis, and Silene dioica. A phylogenetic tree based on internal transcribed spacer data for nuclear rRNA genes of 22 Silene suggested that dioecy in the genus evolved from gynodioecious ancestors (Desfeux et al. 1996). This is consistent with the fact that, whereas the majority of species in the 80 genera in the family Caryophyllaceae are hermaphroditic, many Silene species are gynodioecious and must carry male sterility factors (Defeux et al. 1996). The hermaphroditic and gynodioecious species, S. conica, and S. vulgaris, which are related to the dioecious species, do not have heteromorphic chromosomes. Chromosome heteromorphism therefore reflects de novo evolution of sex chromosomes during the evolution of dioecy in this plant lineage, a relatively recent event within this genus. This gives an opportunity to study processes that occur in young sex chromosomes that are still in earlier stages of their evolution than those of humans or Drosophila.

The evolution of sex chromosomes is believed to involve at least two types of processes. The complete are almost complete genetic degeneration is a dramatic effect that is well known (Lahn and Page 1997; Charlesworth and Charlesworth 2000). Another hypothesized process is the addition to the Y chromosome of genes that are advantageous in males but disadvantageous in females (Charlesworth and Charlesworth 1980; Rice 1997). Because Y chromosomes function in the development of male reproductive organs, new genes may be added to the Y chromosome and may be able to persist despite the forces tending to lead to genetic degeneration. An example of duplicative transfer of autosomal genes to the human Y chromosome is the DAZ gene, which functions in spermatogenesis (Saxena et al. 1996). DAZ reached the Y chromosome by interchromosomal transposition of an autosomal progenitor (Saxena et al. 1996).

We have discovered that a MADS box gene has been duplicated onto the Y chromosome of S. latifolia. The MADS box genes encode transcription factors that share a common DNA-binding domain and represent crucial regulatory genes in plant development, perhaps comparable in importance to the HOX homeobox transcription factor genes in animal development (Ng and Yanofsky 2001; Meyerowitz 2002). The Y-linked MADS box gene evolved from an ancestral autosomal gene through duplication accompanied by increased expression in male reproductive organs. Our finding suggests that duplication of genes onto the Y chromosome may be important in plant, as well as mammalian, Y chromosome evolution, consistent with the theory mentioned above.

Materials and Methods

Plant Material

We used an inbred Silene latifolia line, K1, for the molecular experiments. Seeds of Silene conica, S. diclinis, S. dioica, and other strains of S. latifolia were obtained from the seed banks of the Royal Botanical Gardens (Kew, England), from the botanical gardens at Ulm University (Germany), Regensburg University (Germany), Graz University (Austria), and Portugal University (Portugal), and from the Lake Kawaguchi herb garden (Yamanashi, Japan). Plants were grown in a temperature-controlled chamber at 22°C. Stages of flower buds, including young, early mature, intermediate, and late mature stages, correspond to previously classified stages B1, B2, B3, and B4 plus B5 (Matsunaga et al. 1996).

Cloning of SlAP3 and Orthologs

We designed two sets of degenerate PCR primers, including 5′-CGGAATTCATGAARMGIATIGAIAA-3′ and 5′-CGGGATCCITCIARYTGICBYTCIA-3′ or 5′-CGGGATCCYTCIGCRTCRCAIAGIAC-3′ based on the highly conserved MADS box domain and K domain. The symbols I, R, Y, and B denote inosine, purines (A and G), pyrimidines (C and T), and mixtures without A (C, G, and T), respectively. Hemi-nested PCR amplification was performed using young flower-bud cDNA (five cycles: 94°C for 25 s, 37°C for 2 min, and 72°C for 1 min and 30 cycles: 94°C for 20 s, 55°C for 1 min, and 72°C for 1 min). Amplified fragments were subcloned using a TOPO TA Cloning kit (Invitrogen). The nucleotide sequences were determined using an ABI 3100 genetic analyzer and a BigDye terminator cycle sequencing kit (Applied Biosystems). We obtained full-length cDNAs from screening of our constructed Tripl Ex2 cDNA library (Clontech) or RACE-PCR using SMART RACE cDNA amplification kit (Clontech).

Genomic Distribution

Genomic DNA for southern hybridization was isolated from young leaves using an automatic DNA isolation system PI50 (Kurabo). Preparation of membranes was performed as described previously. Probe preparation, hybridization, and detection were performed using the AlkPhos direct labeling and detection system with CDP-star (Amersham). PCR analyses using genomic DNA and flow-sorted chromosomes were done as described previously (Kejnovsky et al. 2001). The oligonucleotide primers used for the K domains of SlAP3 were as follows: 5′-GTACGATGAGTACCAGAAGA-3′ and 5′-GATCCATGAGGAGATCTCCA-3′. Genomic clones were isolated from a genomic phage library derived from male leaves as described previously (Uchida et al. 2002).

Expression Analyses

Total RNA was isolated from nitrogen-frozen organs using Trizol (Lifetech). Northern hybridization was performed using the Gene Images random-prime labeling and detection system (Amersham). Total RNA was reverse transcribed into cDNA using a first-strand cDNA synthesis kit (Amersham). RT-PCR was performed using Ready-to-Go RT-PCR beads (Amersham) with cDNA. Quantitative RT-PCR was performed using two different systems, the LightCycler (Roche) with LightCycler-DNA master SYBR green I kit (Roche) and the Smart Cycler (Takara) with QuantiTect SYBR green kit (Qiagen). The gene for the GTPase beta subunit (SlGb), which is expressed constitutively in all organs (Matsunaga, unpublished data), was used as an internal standard to estimate the relative expression of mRNA. The relative expression of mRNA for a given tested gene was defined as the mean value that was divided by the mean for SlGb, with the same cDNA used as template. Relative expression values and corresponding standard deviations for the transcripts were calculated from four to six experimental replicates with each of the two real-time PCR systems. The oligonucleotide primer sets used for quantitative RT-PCR were as follows: 5′-GACATGGTGACAGCCATAGCAACA-3′ and 5′-TCACGAGAAGCAGAGACTATCTGT-3′ for SlGb; 5′-GGCATGGAGATCTCCTCATGGATC-3′ and 5′-ATACTGGAGATAACACAGCCTAGT-3′ for SlAP3A; 5′-GGCATGGAGATCTCCTCATGGATC-3′ and 5′-TATATTCGAGACAACATGGCCTGG-3′ for SlAP3Y; and 5′-GGCATGGAGATCTCCTCATGGATC-3′ and 5′-ATATTCGAGACAACATGGCCTAGT-3′ for ScAP3A. Using these primers, only a single fragment was observed in agarose gel electrophoresis after the RT-PCR. The band lengths for SlGb, SlAP3A, SlAP3Y, and ScAP3A, were 330 bp, 313 bp, 310 bp, and 312 bp, respectively. In situ hybridization was performed as described previously (Matsunaga et al. 1996), with an automatic ISH robot AIH-101B (Aloka) using tyramide amplification in the GenPoint system (Dako).

Sequence Diversity and Divergence Analyses

The coding sequences were aligned by eye, with adjustments using BioEdit software (http://www.mbio.ncsu.edu/BioEdit/bioedit.html), and analyses were performed on the aligned sequences using DNAsp version 3.95 (Rozas and Rozas 1999). The average numbers of pairwise differences, _π_s and _π_a, for synonymous and nonsynonymous sites, and their standard deviations were estimated for the samples of 12 S. latifolia SlAP3A and SlAP3Y sequences, each from a different population of origin. The mean pairwise divergence between these sets, in the core and noncore regions (Ng and Yanofsky 2001), and between each of them and the S. conica ScAP3A sequence at silent and replacement sites (_K_s and _K_a), were also estimated, as were the same quantities for the smaller samples of the two other dioecious species (S. dioica, n = 3; and S. diclinis, n = 1). The same software was used for the McDonald-Kreitman tests (McDonald and Kreitman 1991) and Tajima's D statistic (Tajima 1989). To estimate changes in the separate Y-linked and autosomal lineages since the duplication, the “preferred and unpreferred synonymous substitutions” analysis of DNAsp version 3.95 was used with S. conica as the outgroup, assuming parsimony.

Tajima's relative rate test (1993) was done by using parsimony to infer sites fixed within the Y chromosome lineage before the divergence of the three dioecious species, sites fixed in the autosomal lineage, and sites that are unique to the single S. conica sequence. A Neighbor-Joining tree based on the sequence data from all the species was made using MEGA2 (Kumar et al. 2000).

Results and Discussion

Identification of a Y-linked MADS Box Gene with an Autosomal Paralog

Degenerate primers whose sequences were designed from the conserved MADS and K domains were used in RT-PCR utilizing RNA from young S. latifolia male flower buds. Sequencing revealed seven different products. Three sequences were identified as known autosomal MADS box SLM genes (Hardenack et al. 1994; Guttman and Charlesworth 1998). Another sequence showed significant similarity to APETALA3, which in Arabidopsis thaliana functions in defining the floral identity of petals and stamens (Jack et al. 1992). This sequence differs from a previously reported S. latifolia AP3 homolog, SLM3, which functions in the identity of petals and stamens in floral meristems (Hardenack et al. 1994). We therefore named it SlAP3 (S. latifolia AP3). SlAP3 has 78% amino acid identity with an AP3 homolog, CMB2, of Dianthus caryophyllus in the tribe Caryophyllidae (Baudinette et al. 2000). Higher eudicots have two AP3 gene lineages (Kramer, Dorit, and Irish 1998). SLM3 is in the eu-AP3 lineage, whereas SlAP3 and CMB2 fall among genes in the TM6 lineage, most of which are expressed in developing floral organs, rather than in floral meristems. For example, TM6 itself is highly expressed in developing petals, stamens, and carpels but does not play a role in the identity of petals and stamens (Pnueli et al. 1991).

Genomic southern hybridization of SlAP3 with genomic DNA of a pair of male and female parents and four progeny of each sex showed that there are at least two paralogous sequences, one or more apparently autosomal or X-linked, and one present only in males and therefore probably Y-linked (fig. 1_A_). To investigate these loci more fully, we obtained and sequenced full-length homologous cDNAs from a male flower bud library. Twenty-two cDNAs isolated from 2 × 105 recombinants fell into only two different sequence classes. The putative coding and 3′ noncoding sequences of these cDNAs are 92.9% and 85.0% identical, respectively, so we could design PCR primers specific for the two loci. PCR with these primers detected one type of sequence (denoted here by SlAP3A) in both males and females, and a paralog (SlAP3Y) present only in males (fig. 1_A_, lower part), confirming the Y-linkage of SlAP3Y. To determine the chromosomal location(s) of SlAP3A sequences, we performed PCR with flow-sorted X chromosomes and autosomes using primers for the conserved K domains of SlAP3A and SlAP3Y or specific primers for SlAP3A (Kejnovsky et al. 2001), which showed clearly that SlAP3A has no X-linked homolog (fig. 1_B_). Moreover, genomic clones of both SlAP3A and SlAP3Y have five exons and four introns. The SlAP3A genomic clone includes an internal _Hin_dIII restriction site, whereas SlAP3Y has no _Hin_dIII site (Matsunaga, unpublished data). Our results thus indicate that SlAP3 has only two paralogs, SlAP3A and SlAP3Y, in the male genome.

SlAP3A and SlAP3Y Are Functional Duplicates, Evolving Under Selective Constraint

It is very interesting to find a Y-linked locus with no X-linked homolog. One possibility is that this is simply a nonfunctional duplication. This seems unlikely since both paralogs, including SlAP3Y, are present in cDNA, but we have several further pieces of evidence. First, we investigated SlAP3 evolution in the genus and estimated selective constraint on the protein encoded by the duplicates by measuring _K_a and _K_s (the estimated mean numbers of nonsynonymous substitutions per nonsynonymous site and synonymous substitutions per synonymous site) between different sequences. Loss of function of a gene is indicated by ratios of _K_a/_K_s greater than 1 (neutral evolution), and values greater than 0.5 might thus be observed if duplicate genes are compared and one of them has lost its function. These analyses used SlAP3 orthologs from the related hermaphrodite species S. conica and included two dioecious species that are close relatives of S. latifolia. 3′-RACE and 5′-RACE yielded two sets of orthologs from male flower buds, SdAP3A and SdAP3Y from S. dioica and SiAP3A and SiAP3Y from S. diclinis. PCR with specific primers for SdAP3Y and SiAP3Y showed male-specific amplification; thus both these species also have Y-linked SlAP3Y homologs. From the bisexual flower buds of S. conica, only one ortholog, ScAP3A, could be isolated. PCR with primers for ScAP3A, and genomic southern hybridization analysis (data not shown) indicated that the SlAP3 ortholog is a single-copy gene in the S. conica genome. In all comparisons involving sufficient divergence, the _K_a/_K_s ratios of SlAP3A and also SlAP3Y are considerably less than 1 (table 1), suggesting that both SlAP3Y and SlAP3A evolved under selective constraint, that is, that SlAP3Y has retained functionality for most of its evolutionary history (although this test cannot exclude recent loss of function). In support of this conclusion, the 5′ half of the sequence, which encodes the “core region,” including the MADS box (Ng and Yanofsky 2001), has particularly low _K_a/_K_s ratios in both paralogs (using divergence from the S. conica sequence, the values for SlAP3A and SlAP3Y are, respectively, 0.125 and 0.086 for the core versus 0.415 and 0.435 for the remainder of the coding sequence; using the divergence between SlAP3A and SlAP3Y, the two regions' values are 0.276 and 0.487). This differs from the findings in Hawaiian silverswords, where an AP3 gene is present as duplicate copies because the species are tetraploid; in these (unlike their diploid North American relatives) mean _K_a/_K_s ratios were close to 0.5 (Barrier, Robichaux, and Purugganan 2001).

SlAP3A and SlAP3Y Are Expressed Differently During Flower Development

Northern hybridization and RT-PCR analyses using reproductive and vegetative organs of males and females showed that both SlAP3A and SlAP3Y are expressed specifically in reproductive organs, including flower buds and flowers (fig. 2_A_). To determine which regions of the floral organs contained these transcripts, we performed in situ hybridization analysis using as probes the locus-specific 3′ noncoding sequences mentioned above. No transcripts of SlAP3A and SlAP3Y were detected in the second or third whorls of floral meristems of either sex until the stamen primordia emerged. Moreover, levels of both transcripts were low in very young petals and stamen primordia. In contrast, large amounts of both gene transcripts were detected in developing floral organs (fig. 2_B_). These findings are consistent with data for most other TM6 lineage genes, which are reported to be expressed in developing floral organs, rather than floral meristems (Kramer, Dorit, and Irish 1998). In female flower buds, low SlAP3A transcription levels were detected in the ovary walls but were significantly elevated in developing petals, style primordia, and ovules (fig. 2_B_). The growth of stamen primordia is arrested in female flowers and the tissues degenerate before the flowers open (Grant, Hunkirchen, and Saedler 1994). No SlAP3A transcription was detected in arrested stamens (fig. 2_B_). In male flower buds, the carpel primordium is suppressed and later becomes a rudimentary gynoecium (Grant, Hunkirchen, and Saedler 1994), in which SlAP3Y transcription was detected at low levels (fig. 2_B_). The SlAP3Y transcript accumulated at high levels in stamens and petals.

Although SlAP3Y was absent from the female flower buds, a hybridization signal above background was detected in female buds, probably due to cross-hybridization between the SlAP3A and SlAP3Y probes. To more clearly distinguish the SlAP3A and SlAP3Y expression patterns, we therefore performed quantitative PCR with locus-specific primers based on the 3′ noncoding region sequences (figs. 3_A_ and B). Relative expression values and corresponding standard deviations for the transcripts were calculated from at least four experimental replicates with two different real-time PCR systems. SlAP3A and SlAP3Y showed different developmental expression profiles during flower bud maturation. In both male and female flower buds, expression of SlAP3A increased dramatically in early mature buds and then gradually decreased (fig. 3_A_). SlAP3A transcript accumulated abundantly in petals of both male and female buds and was also present at high levels in the females' styles (fig. 3_B_). In contrast, SlAP3Y transcription increased steadily throughout male flower bud maturation (fig. 3_A_), with high transcript levels in petals and higher transcript levels in stamens (fig. 3_B_).

Gene duplications are a source of evolutionary novelties, including new gene functions and expression patterns (Ohno 1970). Our results suggest that both duplicates in the dioecious Silene species have escaped silencing by degenerative mutations. The expression data suggest that the duplicate copy retains only some of the original functions and tissue expression locations. These changes in expression may have involved mutations in the genes' regulatory regions or in the coding regions (Force et al. 1999). Estimated sequence changes in the Y-linked versus autosomal gene lineages since the duplication do not differ significantly (the numbers were, respectively, nine versus seven synonymous changes and seven versus 11 nonsynonymous changes). Again, unlike Hawaiian silverswords (Barrier, Robichaux, and Purugganan 2001), there is no evidence for accelerated subsitution in either Silene duplicate and thus no suggestion that either is evolving a new function. This is supported by nonsignificant McDonald-Kreitman tests (McDonald and Kreitman 1991).

The evolution of the SlAP3 duplicates may thus have involved mainly regulatory mutations changing the temporal expression of SlAP3A, so that expression of SlAP3Y has changed from petal-specific to largely anther expression. Even without changes in the coding regions, quantitative subfunctionalization is possible, resulting from mutations reducing the expression of both copies (Force et al. 1999). To test for this possibility, we did quantitative RT-PCR with primers from identical regions of ScAP3A, SlAP3A, and SlAP3Y (fig. 3_C_). If SlAP3A and SlAP3Y are both expressed in petals in the same manner as the ancestral gene, the total transcript level in male flowers should be twice that in the bisexual flower of S. conica. However, the level of SlAP3A plus SlAP3Y in petals of male flowers was similar to the level of the single-copy ScAP3A, whereas SlAP3A in female flowers' petals was lower (fig. 3_C_).

The Duplication Occurred After the Evolution of the Sex Chromosomes

Given the evidence for altered gene expression, it is of interest to ask when the duplication that created the autosomal and Y-linked paralogs occurred, relative to the origin of the sex chromosomes. This can be estimated from the synonymous nucleotide divergence in the coding regions of the SlAP3 homologs (table 1), assuming that synonymous substitutions are close to neutral. Divergence between the autosomal and Y-linked paralogs is lower than divergence of either of them from ScAP3A (table 1), suggesting that the duplication event occurred after the divergence of S. conica and the three dioecious species (fig. 4). Tajima's relative rate test (1993) was nonsignificant for the entire sequence (800 nucleotides) or for the coding sequence, for which the alignment is more certain. In the Neighbor-Joining tree, there was 99% bootstrap support for the branch of the tree that includes all the Y sequences and for the autosomal sequences (data not shown). Under the alternative hypothesis that S. conica had two loci and lost one of them, whereas the dioecious species retained both (one remaining autosomal and the other being on the chromosome that evolved to become the Y), the divergence values would be expected to be very different from this. Sequence divergence should then be lowest between the S. conica ScAP3A and its ortholog in the dioecious species, whereas divergence between ScAP3A and the nonorthologous sequence should be much larger, since the duplication that created the two genes must predate the split between the ancestors of S. conica and the dioecious species. However, both sequences in the dioecious species are roughly equally diverged from ScAP3A, which rules out this possibility. Moreover, the _K_s values for SlAP3A versus SlAP3Y are consistent with those for the two known S. latifolia X-linked and Y-linked gene pairs (Atanassov et al. 2001), suggesting that the duplication occurred soon after the evolution of the sex chromosomes.

SlAP3Y Acquired a Male Function Despite Degenerative Forces on the Y Chromosome

SlAP3Y is expressed almost exclusively in developing stamens, which strongly suggests that it functions in the maturation of anthers and pollen grains (figs. 2_B_ and 3_B_). SlAP3Y has a different function in male fertility from that of the autosomal SlAP3A, which appears to have preserved ancestral functions expressed during petal development, that is, SlAP3Y probably acquired a new function in anther maturation after its duplication to the Y chromosome. This presumably accounts for its having maintained function, despite the degenerative forces acting on nonrecombining Y chromosomes (Charlesworth and Charlesworth 2000). Processes involved in the genetic degeneration have been inferred from evidence for a reduction in effective population size for Y-linked genes. Assuming equilibrium under neutrality, Y-linked genes should have one quarter of the diversity of autosomal loci. Significantly lower diversity than this neutral expectation has been found for certain genes on the neo-Y chromosome of a fruitfly, Drosophila miranda, based on lower levels of synonymous nucleotide variability (Bachtrog and Charlesworth 2002). In S. latifolia, the Y-linked genes SlY1 and SlY4 also have lower nucleotide variability than predicted (Filatov et al. 2000; Laporte, Filatov, and Charlesworth, unpublished data). However, the only autosomal locus so far surveyed also had low variability, and analysis of its variants suggests that diversity may have been reduced by a recent selective sweep (Filatov et al. 2001), so that it is uncertain whether Y chromosomal variability is low or the X-linked genes have unusually high diversity.

We have compared the species-wide nucleotide diversity of SlAP3Y with that of the autosomal SlAP3A using sequences of these genes from 12 different populations. First, the autosomal locus diversity is consistent with that predicted from the previously studied X-linked genes, making an elevated X-linked diversity unlikely. Comparisons with autosomal genes are not possible at present, since, as just mentioned, the only other such gene cannot be assumed to be at equilibrium, but may have recently lost diversity. Diversity of X-linked loci could, however, be used to predict the expected neutral diversity value for autosomal genes. Two loci have been surveyed. The species-wide synonymous site π values were about 0.027 for SlX1 (Filatov et al. 2001), and the very high value of 0.059 for SlX4, possibly due to introgression from S. dioica (Laporte, Filatov, and Charlesworth, unpublished data). These results suggest an expected autosomal value of about 3% to 7%, and 1% to 2% for Y chromosomal genes. Both these are considerably higher than our observed values (note, however, that the samples are comparable, although not from the same set of populations). If these high values are typical, which is not certain, given the high variance of such estimates, our autosomal locus has lower than the predicted diversity. Even based on the low SlAP3A value, SlAP3Y diversity, measured as either the mean pairwise proportion of sites differing between alleles (π) or from the number of segregating sites (𝛉) was six to nine times lower than the SlAP3A value (table 2); based on the standard errors of the 𝛉 values, this is just significantly below the predicted value of four times lower. Thus, as for the other Y-linked genes in this species, we conclude the diversity of SlAP3Y is smaller than that of SLAP3A to an extent greater than expected. The effect is less than for SlY1, but it again suggests that selective events at other Y-linked loci may be reducing diversity, as expected if this Y chromosome is undergoing a process of degeneration (Charlesworth and Charlesworth 2000).

Duplications to the Y chromosome may be a regular event in the evolution of sex chromosome systems, allowing the accumulation of genes that are advantageous in males but disadvantageous in females (Charlesworth and Charlesworth 1980; Rice 1997). Such a hypothesis is plausible for SlAP3Y, with its increased stamen expression. Fixation of an interchromosomal transposition would be a unique event, which would initially completely eliminate Y-linked diversity and might potentially be detectable from a pattern of excess low-frequency synonymous variants, which is the signature of a selective sweep associated with the recent spread of an advantageous mutation (Tajima 1989). Such a result would be very interesting because it would support the hypothesis of selection promoting the spread of the Y-linked duplication. However, the SlAP3Y diversity is too low to use its variants in this test. Moreover, we estimate that the event occurred too long ago for any effects of a selective sweep to be detectable, even if one occurred. It seems more likely that the low SlAP3Y diversity is due to currently operating causes, most likely processes involved in Y chromosome degeneration, rather than to such a selective sweep.

Interestingly, in contrast to the testis-specific expression of DAZ, SlAP3Y retains some autosomal ancestral expression in petals (as well as its higher anther expression). The extent of the transposed genomic region that includes SlAP3Y is not yet known, but it probably encompasses not only introns and exons but also the promoter region. The changed SlAP3Y male function may have been caused, in part, by degenerative mutations in the promoter, leading to reduced petal expression. Our results suggest that the acquisition of autosomal genes is an important component of plant Y chromosomes. If so, it is probably important in Y chromosome evolution in general, which has until now been a purely speculative idea.

Supplementary Material

The sequences of the SlAP3A, SlAP3Y, SdAP3A, SdAP3Y, SiAP3A, SiAP3Y, and ScAP3A cDNA have been deposited in GenBank. Accession numbers are AB090863, AB090864, AB090865, AB090866, AB090867, AB090868, and AB090869, respectively.

1

Present address: Department of Biotechnology, Graduate School of Engineering, Osaka University, Osaka, Japan.

Brandon Gaut, Associate Editor

Fig. 1.

Evidence for the chromosomal locations of the two SlAP3 paralogs. (A) The upper panel shows the genomic southern hybridization analysis of SlAP3. Genomic DNA was digested with _Hin_dIII. The SlAP3A genomic clone includes an internal _Hin_dIII restriction site, whereas SlAP3Y has no _Hin_dIII site. The figure shows the results from two parents (♂1 and ♀1: male and female parents) and some male and female offspring (♂ and ♀: 2 to 5), hybridized with the K domain of SlAP3. Based on the presence of a _Hin_dIII restriction site within the sequences, the expected autosomal pattern has two hybridizing fragments, whereas a single fragment represents the sequence present in the male parent and male progeny. The middle and lower panels show the electrophoretic analysis of PCR products obtained with the _SlAP3A_-specific and _SlAP3Y_-specific primers, respectively. (B) PCR analyses on flow-sorted chromosomes. The PCR products were amplified with the primers for the K domain of _SlAP3_-specific, _SlAP3A_-specific, or _MROS3_-specific primers using female (♀) and male (♂) genomic DNA, no chromosomes (0), flow-sorted autosomes (A), or X chromosomes (X) as templates. The primers for the K domain and SlAP3A amplify 1,094-bp and 411-bp fragments, respectively. The _MROS3_-specific primers amplify 433-bp fragments from both autosomal and X-linked paralogs (Kejnovsky et al. 2001)

Fig. 2.

Expression analyses of SlAP3A and SlAP3Y. (A) The first panel shows northern hybridization analysis of SlAP3. Each lane contained 20 μg of total RNA from male and female organs hybridized with SlAP3. Panels 2 to 4 show RT-PCR analyses using specific primers for SlAP3A, SlAP3Y, and SlGb, respectively. PCR products were amplified using 1 μg of the RNA that was used in the northern blot. (B) In situ hybridization of SlAP3A (left) and SlAP3Y (right) in longitudinal sections of young female and male flower buds, respectively. The hybridization signals appear reddish brown. G = suppressed gynoecium; O = ovary; P = petal; Se = sepal; St = suppressed stamen; T = stamen; Y = style

Fig. 3.

Quantitative analyses of the SlAP3A, SlAP3Y, and ScAP3A transcripts. Bars represent normalized relative expression values. The blue, pink, and green bars represent the relative expression levels in male, female, and bisexual flower buds, respectively. The data are shown as mean ± SEM, where n = 4 to 6. (A) Temporal gene-expression patterns during the development of flower buds. (B) Spatial gene-expression patterns in developing floral organs. (C) Quantitative comparisons of gene expression between the petals of male, female, and bisexual flower buds. “Male” designates expression in petals of SlAP3A plus SlAP3Y, and “female” and “bisexual” represent the expression levels in petals of SlAP3A and ScAP3A, respectively

Fig. 4.

Schematic representation of the evolutionary history of SlAP3. Duplication of the ancestral APETALA3 (white arrowhead) produced the eu-AP3 and TM6 lineages. In the TM6 lineage, duplication and transposition to the Y chromosome (black arrowhead) occurred after the evolution of the sex chromosomes

Table 1

Comparison of Sequence Divergence Between Paralogs and Orthologs of SlAP3.

Table 1

Comparison of Sequence Divergence Between Paralogs and Orthologs of SlAP3.

Table 2

Nucleotide Diversity for Nonsynonymous and Synonymous Sites of the Autosomal and Y-Linked Paralogs.

Table 2

Nucleotide Diversity for Nonsynonymous and Synonymous Sites of the Autosomal and Y-Linked Paralogs.

Financial support was provided by the Ministry of Education, Sports and Culture of Japan (S.M. and S.K.), Grant Agency of the Czech Republic (E.K., B.V., and J.D.: No. 521/02/0427 and 204/02/0417), and the Natural Environment Research Council of Great Britain (D.C.).

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