* Department of Integrated Biosciences, Graduate School of Frontier Sciences, University of Tokyo, Chiba, Japan
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan
Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic
Institute of Experimental Botany, Academy of Sciences of the Czech Republic, Olomouc, Czech Republic
|| Institute of Cell, Animal and Population Biology, University of Edinburgh, Edinburgh, United Kingdom
Correspondence: E-mail: sachi{at}bio.eng.osaka-u.ac.jp.
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Abstract |
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Key Words: dioecious plant Y chromosome sex differentiation MADS box gene subfunctionalization duplication
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Introduction |
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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.
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Materials and Methods |
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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, s and
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 (Ks and Ka), 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).
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Results and Discussion |
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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. 1A). 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 x 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. 1A, 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. 1B). Moreover, genomic clones of both SlAP3A and SlAP3Y have five exons and four introns. The SlAP3A genomic clone includes an internal HindIII restriction site, whereas SlAP3Y has no HindIII site (Matsunaga, unpublished data). Our results thus indicate that SlAP3 has only two paralogs, SlAP3A and SlAP3Y, in the male genome.
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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. 3C). 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. 3C).
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 Ks 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.
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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 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 (
) or from the number of segregating sites (
) was six to nine times lower than the SlAP3A value (table 2); based on the standard errors of the
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).
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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.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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Brandon Gaut, Associate Editor
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