Laboratoire de Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, Lyon, France;
Institute of Genetic Engineering, Kostinbrod, Bulgaria;
Laboratoire de Biochimie et Physiologie Moléculaire des Plantes, Université Montpellier II, Montpellier, France;
Institute of Cell, Animal, and Population Biology, University of Edinburgh, Edinburgh, Scotland
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Abstract |
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Introduction |
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Sex chromosomes are believed to be evolutionarily derived from a pair of ordinary autosomes (Bull 1983
; Ellis 1998
). It is presumed that sex was initially genetically determined by a system in which the male was heterozygous at two loci. Classical cytogenetic and genetic work showed that at least two sex-determining factors (female inhibition and male activation) are indeed involved in white campion (Westergaard 1958
; Farbos et al. 1999
; Lardon et al. 1999
). Subsequent to this initial stage, meiotic recombination between the proto-sex chromosomes (or parts of them) was suppressed. Thus, the Y chromosome of many species, including S. latifolia, in which pairing is confined to a terminal region (Farbos et al. 1999
), differs from other chromosomes in that it does not recombine along the majority of its length, in addition to its being present only in the male sex in a permanent haploid condition.
Arrest of X-Y recombination appears to be the critical event in the evolution of sex chromosomes and is expected to lead to subsequent genetic events, including degeneration of Y-linked loci (Charlesworth 1996
; Mitchell 2000
) and dosage compensation, for which there is some evidence in plants (Vyskot et al. 1993
; Siroky et al. 1994
; Siroky, Castiglione, and Vyskot 1998
). In the much better studied human Y chromosome, Lahn and Page (1999)
analyzed 19 genes with homologs on both the X and the Y chromosomes and estimated their divergence times from nucleotide divergence values at silent sites. They proposed that human Y chromosome evolution involved four inversions, each separately suppressing X-Y recombination without disturbing the gene order on the X chromosome. These events are estimated to have spanned a timescale of 240300 Myr.
In addition to the sex-determining gene SRY, three groups of genes have been identified in the nonrecombining region of the human Y chromosome (Delbridge and Graves 1999
; Lahn and Page 1999
). One group contains genes shared between the X and Y chromosomes. This group includes housekeeping genes that are ubiquitously expressed and escape X-inactivation (both X and Y copies are expressed and thus produce two doses of the corresponding gene product). The other two groups, however, differ between the X and Y chromosomes. One category contains testis-specific genes with widespread expression of the X-linked homolog (dosage compensation of the X copy allows the Y copy to acquire male-enhancing functions). The final category contains testis-specific multicopy genes with no homologs on the X chromosome (these are thought to have originated through retrotransposition from autosomes). These categories reflect the diminishing contribution of Y-chromosomal alleles to various developmental processes (Mitchell 2000)
.
In white campion, sexual dimorphism and its control by an XY sex chromosome system probably evolved relatively recently, as other species in the genus are hermaphroditic or gynodioecious (Desfeux et al. 1996
). Based on sequence divergence for the internal transcribed spacer (ITS) sequences of ribosomal DNA (Desfeux et al. 1996
), using accepted rates of silent site substitutions in plants (Gaut 1998
), we estimate that in the genus Silene, the sex chromosomes probably evolved about 20 MYA. Plant sex chromosomes therefore represent a unique opportunity to study early steps of sex chromosome evolution. For such work, it is clear that molecular characterization of sex-linked loci is required. We have therefore started a search for sex-linked loci in S. latifolia. The first Y-linked gene described, SlY1 (Delichère et al. 1999
), has a very similar X-chromosomal copy, and molecular evolutionary studies of these loci suggest reduction of the Y-chromosome effective population size, showing that some selective processes must affect this plant Y chromosome (Filatov et al. 2000)
.
In this paper, we describe the characterization of a novel Y-linked gene from S. latifolia, SlY4. Like SlY1 (Delichère et al. 1999
), SlY4 also has a homolog on the X chromosome, SlX4. SlY4 and SlX4 are both expressed in all of the tissues tested and are predicted to encode fructose-2,6-bisphosphatases, the sequences of which are reported for the first time in plants. SlY4/SlX4 and SlY1/SlX1 are the first reported active genes located on plant sex chromosomes. Both the Y-linked genes were isolated from a cDNA library, and their expression was detected in several tissues. In addition, analysis of the rates of synonymous substitutions (Ks) and nonsynonymous substitutions (Ka) for each pair of sex-linked loci revealed selective constraints, suggesting that they encode functional proteins. Furthermore, this analysis indicates that the two loci characterize Y-chromosome regions that have ceased recombining at different times during the evolution of sex chromosomes.
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Materials and Methods |
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Screening of cDNA and Genomic Libraries
The screening of the cDNA library of male flowers at premeiotic and meiotic stages with the Y-derived probe was as described by Delichère et al. (1999)
. The genomic library of male plants (a kind gift from P. Gilmartin) was constructed into Lambda FIX II (Stratagene). One million plaques were transferred onto Hybond N+ nylon membranes (Amersham) and were hybridized with the two cDNAs, SlY1 and SlY4. DNA was extracted from the phages containing inserts hybridizing to the probes, and restriction fragments were subcloned into pBluescript plasmid (Stratagene).
Genomic Southern Blot Analysis and Northern Blot Analysis
Genomic Southern blot analysis and Northern blot analysis protocols were as described by Delichère et al. (1999)
.
PCR
Rapid amplification of cDNA ends (RACE)-PCR and reverse-transcribed (RT)-PCR were as described by Delichère et al. (1999)
. For RACE-PCR, the primers 95S1 (CCACTGGGAAGGTTGCCCTCGTTCT) and 95S3 (CCGAAGCTCCATTAGCGAACCGAATAC) were used for the 3' amplification, and the primer 95AS4 (GACGTCGGCTCCTACGGACAGTT) was used for the 5' amplification. The gene-specific primers used for both RT-PCR and PCR from genomic DNA for SlY4 were CAACCTGACTTCTCCGCTCCTTCTGG and CAACATGAGCTCCTCGTGAGCACGGCG, and those for SlX4 were CCACTGGGAAGGTTGCCCTCGTTCT and CCGAAGACAGTAAACCGTCAACCCAACC. For the SlY4-specific set of primers, the conditions were as follows: 3 x (30 s at 95°C, 30 s at 65°C, 2 min at 72°C); 3 x (30 s at 95°C, 30 s at 62°C, 2 min at 72°C); 27 x (30 s at 95°C, 30 s at 59°C, 2 min at 72°C). For the SlX4-specific set of primers, the conditions were as follows: 3 x (30 s at 95°C, 30 s at 68°C, 2 min at 72°C); 3 x (30 s at 95°C, 30 s at 65°C, 2 min at 72°C); 27 x (30 s at 95°C, 30 s at 62°C, 2 min at 72°C).
Cloning and Sequencing
Cloning and sequencing were done as described by Delichère et al. (1999)
.
Sequence Comparisons
Sequence data were initially analyzed using BLAST and BESTFIT (GCG7.3 version) using the blosum62.cmp matrix. Multiple protein sequence alignments were performed using the program MEGALIGN (DNASTAR). To assess amino acid sequence conservation, Ka and Ks values were calculated with the MEGA program (Kumar et al. 2000)
using P-distances (the proportion of sites at which the two sequences compared are different, without correction for transition-vs.-transversion bias in substitutions). No correction was made for multiple substitutions at the same site, which is conservative for our purposes, as it underestimates silent substitutions between highly diverged sequences and therefore underestimates the degree of conservation of amino acids (Li 1997
). For Silene conica, portions of the genes orthologous to SlX4/SlY4 and SlX1/SlY1 were sequenced from genomic DNA and analyzed after removal of intron sequences. In both cases, the PCR products were single sequences, suggesting that only one homolog of both genes is present in the S. conica genome.
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Results |
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Analysis of SlY4 and SlX4 Transcripts
In order to identify complete cDNAs for both SlY4 and SlX4, we performed RACE-PCR and RT-PCR experiments. The longest cDNA obtained was 1,713 bp (excluding the polyA tail). Given the estimated size of about 1,750 bases from Northern blot analysis shown in figure 2A,
we believe that this transcript is nearly or entirely complete. Consistent with this view, the 5' region of the cDNA contains stop codons and is rich in CT motifs (9.8% in the 194 bp of the putative 5' untranslated region vs. 4% in the open reading frame), as previously observed in 5' untranslated regions of other cDNAs from S. latifolia (unpublished data). These data suggest that our cDNA contains a complete open reading frame.
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A Northern blot analysis of total RNA is shown in figure 2A. Transcripts homologous to the cDNA are detected in all of the tissues tested: premeiotic (stage 1) and postmeiotic (stage 2) flower buds from both males and females, and leaves, shoots, stems, and seedlings from mixtures of male and female plants. To test for tissue-specific expression patterns of SlY4 and SlX4, RT-PCR experiments were performed using the gene-specific primer sets used on genomic DNA (fig. 2B ). The results, shown in figure 2C, demonstrate that both SlY4 and SlX4 are expressed in all of the tissues in which Northern blotting detected transcripts (fig. 2A ). In female plants, SlX4 was also detected in pre- and postmeiotic flower buds. These results suggest that both SlY4 and SlX4 are ubiquitously expressed in S. latifolia.
SlY4 and SlX4 May Encode Fructose-2,6-Bisphosphatases
The polypeptide sequences deduced from the SlY4 and SlX4 cDNAs (respectively, 422 and 425 amino acids long) were compared with sequences in the databases. The two highest protein similarity scores were obtained with two predicted proteins from Arabidopsis thaliana: the first one (SPTNEW BAB01224, on chromosome 3) shared 65.8% identity with SlY4 and 68.1% with SlX4. The second (SPTNEW AAF78490, on chromosome 1) shared 64.6% identity with SlY4 and 65.4% with SlX4. Protein sequence alignments showed significant homology between SlY4, SlX4 and the two homologous proteins from A. thaliana, and the bisphosphatase domain of the 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatases (bifunctional 6PF2-K/Fru-2,6-P2ases) from animals (42.5%48.3% similarity, based on 133 amino acids). The important residues were conserved, indicating that the plant genes analyzed probably encode monofunctional fructose-2,6-bisphosphatases. Surprisingly, these fructose-2,6-bisphosphatases have higher similarity to animal than to plant bifunctional enzymes.
Comparative Analysis of the Two X- and Y-Linked Loci
In order to investigate the genomic organization of the two sex-linked loci characterized so far, SlY4/SlX4 and the previously described SlY1/SlX1 (Delichère et al. 1999
), we used the cDNAs as probes on a male genomic library. A genomic clone was isolated for each gene. The entire nucleotide sequence was determined for SlX1, and almost all of it was determined for SlY1. For SlX4, the 5' part of the cDNA was not present in our genomic clone, so the first exon is missing. Finally, the first intron of the SlY4 genomic clone was not sequenced. Alignment with the corresponding cDNA sequences allowed the localization of the introns.
The genomic structures of the two loci are shown in figure 3 . SlY1 and SlX1 were very similar in structure. Both contained 14 introns which had the same locations and were highly conserved in both size and sequence: the average identity was 96.8%, with a range of 93.2%99%. The genomic structures of the SlY4 and SlX4 genes were also similar, with both genes having at least two long introns in the same positions. The intron sizes differed considerably, however. The second intron of SlX4 was 2,853 bp long, while that of SlY4 was 4,167 bp long, and they shared no significant identity, even locally.
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Discussion |
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The sex chromosomes of S. latifolia evolved recently (Desfeux et al. 1996
), whereas genetic degeneration probably requires large amounts of evolutionary time. Recent evolution may therefore increase the chance of finding Y-linked genes that have detectable X homologs and are transcribed and encode functional proteins. Our finding of two such genes does not necessarily reflect a low level of degeneration for this Y chromosome, but may simply represent a bias due to the fact that only transcribed genes would be detected by our approach. To determine what proportion of X-linked genes remain as expressed loci on the Y chromosome, it will be necessary to test an unbiased set of X-linked genes. To date, only one such gene (MROS3) has been tested, and its Y-linked counterpart was found to have degenerated (Guttman and Charlesworth 1998
).
The fact that both of the Y-linked genes have low Ka/Ks ratios in both inter- and intraspecies comparisons indicates that amino acid replacements are much less frequent than silent changes, given the relative numbers of silent and replacement sites in the sequences. This is good evidence that both the SlY1 and the SlY4 genes have encoded functional proteins during most or all of the period since their evolutionary divergence from their respective X homologs and that their coding sequences are evolving as expected for genes encoding proteins with substantial natural selection against amino acid sequence changes. The possibility that the Y-linked genes have only recently ceased to encode functional proteins is disproved by the observation that the SlY4 and SlX4 are much more diverged from one another than are SlY1 and SlX1. It is highly unlikely that the SlY1 and SlY4 genes both lost their function recently, despite their very different divergence times. In addition, all of these genes have been shown to be transcribed. The sequences of these genes give no other indications of loss of function, such as stop codons or frameshifts.
Cessation of Recombination in the S. latifolia Y Chromosome
The characterization of these first two X- and Y-linked loci allows us to compare the evolution of plant sex chromosomes with those of mammals. The human X and Y chromosomes have been proposed to have at least four "evolutionary strata" with different amounts of divergence (Lahn and Page 1999
). In S. latifolia, the divergence between SlY4 and SlX4 is much greater than that between the SlY1 and SlX1 genes. Silent-site divergence between S. latifolia SlY4 and SlX4 is about 18%, compared with 4% between SlY1 and SlX1, and amino acid differences between SlY4 and SlX4 are also much greater than those between SlY1 and SlX1 (table 1 ). The SlY4/SlX4 silent-site divergence is similar to the estimated value of about 19% between the X-linked MROS3 gene and the degenerated Y-linked copy, which has been evolving in a neutral manner (Guttman and Charlesworth 1998
). However, this finding is based on only a short sequence, as only a 159-bp region has high homology between the X- and Y-linked homologs. Assuming a synonymous molecular clock with a rate of about 0.6%/Myr (Gaut 1998
), we can then estimate that SlY4 and SlX4, and also the MROS3-X and MROS3-Y gene pair, stopped recombining about 15 MYA. This is consistent with the age of this sex chromosome system as estimated from ITS sequences (Desfeux et al. 1996
), but it is much longer ago than the estimate for SlY1 and SlX1 (3.3 Myr). A molecular clock with a constant rate for synonymous sites is, of course, an approximation, but the more than fourfold smaller synonymous-site divergence between SlY1 and SlX1 compared with the X- and Y-linked members of the other two gene pairs currently available suggests a difference in their divergence times. Out of 14 genes compared between A. thaliana and Arabidopsis lyrata (mean Ks = 0.21), the least diverged gene has 42% of the Ks value of the most diverged (Lagercrantz and Axelsson 2000)
. As for human XY-linked genes, the plant sex-linked loci analyzed therefore probably ceased recombining at very different times in the evolutionary history of the sex chromosomes. This conclusion should be tested further if additional sex-linked loci can be discovered in this plant.
Genes on an X/Y chromosome pair will start diverging as soon as they become isolated from one another by suppression of recombination. This suppression could have progressed along the sex chromosomes during evolution by successive inversions, as has been proposed for the human sex chromosomes (Lahn and Page 1999
). If this hypothesis is true, SlX1 should be closer than SlX4 to the pseudoautosomal region. An alternative is that the chromosome region containing SlX1 could have been translocated relatively recently onto both members of an X/Y chromosome pair that already carried diverged SlY4/SlX4 genes, similar to the XAR region that has been proposed for mammalian sex chromosomes (Graves 1995
). For either of these hypotheses, the results presented here identify two distinct "strata" within the Y chromosome according to Ks values found for the X- and Y-chromosome gene pairs (Lahn and Page 1999
).
Where are these strata located on the Y chromosome? Available results from Y deletion mapping studies in sexual mutants suggest that the two potential strata identified in this work, carrying SlY1 and SlY4, probably localize somewhere along the q-arm of the Y chromosome outside the pseudoautosomal region. Additional genes located on these plant sex chromosomes are needed to improve the resolution of these first results and to determine more precisely how sex chromosomes have evolved in the plant kingdom.
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Supplementary Material |
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Acknowledgements |
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Footnotes |
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1 Present address: School of Biosciences, University of Birmingham, Edgbaston, Birmingham, England.
Abbreviations: Ka, nonsynonymous substitution rate; Ks, synonymous substitution rate; RT-PCR, reverse-transcribed PCR; SlY1/SlX1, Silene latifolia Y/X-chromosome gene 1; SlY4/SlX4, Silene latifolia Y/X-chromosome gene 4.
Keywords: plant sex chromosomes
dioecy
Silene latifolia
molecular evolution
Address for correspondence and reprints: Françoise Monéger, Laboratoire de Reproduction et Développement des Plantes, Ecole Normale Supérieure Lyon, 46 Allée d'Italie, 69364 Lyon cedex 07, France. francoise.moneger{at}ens-lyon.fr
.
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