Molecular and evolutionary analysis of a plant Y chromosome.

Plants have evolved a great diversity of sex determination systems. Among these, the XY system, also found in mammals, is one of the most exciting since it gives the opportunity to compare the evolution of sex chromosomes in two different kingdoms. Whereas genetic and molecular mechanisms controlling sex determination in drosophila and mammals, have been well studied, very little is known about such processes in plants. White campion (Silene latifolia) is an example of plant with X and Y chromosomes. What is the origin of the X and Y chromosomes? How did they evolve from a pair of autosomes? In our laboratory, we have isolated the first active genes located on a plant Y chromosome. We are using them as markers to trace the origin and evolution of sex chromosomes in the Silene genus.     

The widespread nature of Pack-TYPE transposons reveals their importance for plant genome evolution

Pack-TYPE transposable elements (TEs) are a group of non-autonomous DNA transposons found in plants. These elements can efficiently capture and shuffle coding DNA across the host genome, accelerating the evolution of genes. Despite their relevance for plant genome plasticity, the detection and study of Pack-TYPE TEs are challenging due to the high similarity these elements have with genes. Here, we produced an automated annotation pipeline designed to study Pack-TYPE elements and used it to successfully annotate and analyse more than 10,000 new Pack-TYPE TEs in the rice and maize genomes. Our analysis indicates that Pack-TYPE TEs are an abundant and heterogeneous group of elements. We found that these elements are associated with all main superfamilies of Class II DNA transposons in plants and likely share a similar mechanism to capture new chromosomal DNA sequences. Furthermore, we report examples of the direct contribution of these TEs to coding genes, suggesting a generalised and extensive role of Pack-TYPE TEs in plant genome evolution.     

Duplicative transfer of a MADS box gene to a plant Y chromosome

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.     

Semi-automatic laser beam microdissection of the Y chromosome and analysis of Y chromosome DNA in a dioecious plant, Silene latifolia

Silene latifolia has heteromorphic sex chromosomes, the X and Y chromosomes. The Y chromosome, which is thought to carry the male determining gene, was isolated by UV laser microdissection and amplified by degenerate oligonucleotide-primed PCR. In situ chromosome suppression of the amplified Y chromosome DNA in the presence of female genomic DNA as a competitor showed that the microdissected Y chromosome DNA did not specifically hybridize to the Y chromosome, but hybridized to all chromosomes. This result suggests that the Y chromosome does not contain Y chromosome-enriched repetitive sequences. A repetitive sequence in the microdissected Y chromosome, RMY1, was isolated while screening repetitive sequences in the amplified Y chromosome. Part of the nucleotide sequence shared a similarity to that of X-43.1, which was isolated from microdissected X chromosomes. Since fluorescence in situ hybridization analysis with RMY1 demonstrated that RMY1 was localized at the ends of the chromosome, RMY1 may be a subtelomeric repetitive sequence. Regarding the sex chromosomes, RMY1 was detected at both ends of the X chromosome and at one end near the pseudoautosomal region of the Y chromosome. The different localization of RMY1 on the sex chromosomes provides a clue to the problem of how the sex chromosomes arose from autosomes.     

SlY1, the first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein.

Unlike the majority of flowering plants, which possess hermaphrodite flowers, white campion (Silene latifolia) is dioecious and has flowers of two different sexes. The sex is determined by the combination of heteromorphic sex chromosomes: XX in females and XY in males. The Y chromosome of S.latifolia was microdissected to generate a Y-specific probe which was used to screen a young male flower cDNA library. We identified five genes which represent the first active genes to be cloned from a plant Y chromosome. Here we report a detailed analysis of one of these genes, SlY1 (S.latifolia Y-gene 1). SlY1 is expressed predominantly in male flowers. A closely related gene, SlX1, is predicted to be located on the X chromosome and is strongly expressed in both male and female flowers. SlY1 and SlX1 encode almost identical proteins containing WD repeats. Immunolocalization experiments showed that these proteins are localized in the nucleus, and that they are most abundant in cells that are actively dividing or beginning to differentiate. Interestingly, they do not accumulate in arrested sexual organs and represent potential targets for sex determination genes. These genes will permit investigation of the origin and evolution of sex chromosomes in plants.     

Preservation of the Y transcriptome in a 10-million-year-old plant sex chromosome system

Classical genetic studies discovered loss of genes from the ancient sex chromosome systems of several animals (genetic degeneration), and complete genome sequencing confirms that the heterogametic sex is hemizygous for most sex-linked genes. Genetic degeneration is thought to result from the absence of recombination between the sex chromosome pair (reviewed by [1]) and is very rapid after sex chromosome-autosome fusions in Drosophila [2-4]. Plant sex chromosome systems allow study of the time course of degeneration, because they evolved from a state wholly without sex chromosomes (rather than after a large genome region fused to a preexisting sex chromosome), and, in several taxa, recombination stopped very recently. However, despite increasing genetic and physical mapping of plant nonrecombining sex-determining regions [5-8], it remains very difficult to discover sex-linked genes, and it is unclear whether Y-linked genes are losing full function. We therefore developed a high-throughput method using RNA-Seq to identify sex linkage in Silene latifolia. Recombination suppression between this plant's XY sex chromosome pair started only about 10 million years ago [9]. Our approach identifies several hundred new sex-linked genes, and we show that this young Y chromosome retains many genes, yet these already have slightly reduced gene expression and are accumulating changes likely to reduce protein functions.     

The Y chromosome-specific STS marker MS2 and its peripheral regions on the Y chromosome of the dioecious plant Silene latifolia.

Sex determination in Silene latifolia uses the XX/XY system. The recent evolution of dioecy in S. latifolia provides a unique opportunity to study the early stages of Y chromosome evolution. However, the current Y chromosome map still contains many large gaps with no available markers. In this study, a sequence tagged site (STS) marker, MS2, was isolated and mapped to the same locus as L8 on the Y chromosome. To investigate the peripheral regions of MS2, a bacterial artificial chromosome (BAC) library was constructed from a male plant, and the BAC clone containing MS2 (MS2-9d12F) was isolated from 32 640 clones with an average insert size of 115 kb. A 109-kb insert of the BAC clone was analyzed. BLASTX analysis showed 11 sequences similar to some known proteins, most of which are retrotransposon-like elements. The ORF Finder predicted 9 ORFs within MS2-9d12F. RT-PCR analyses revealed that only 4 of the 9 predicted ORFs are expressed in both male and female plants. These 4 ORFs are candidates for genes having counterparts on both the X and Y chromosomes. Dot-matrix plot analysis and a BLASTN search revealed LTR-like sequences close to the retrotransposon-like elements and high similarity to 3 known genomic sequences of S. latifolia. These results suggest an accumulation of retrotransposons and segmental duplications in peripheral regions of MS2 during the early stage of sex chromosome evolution.     

Sexual ambiguity and a non-fluorescent Y chromosome

The personal experience in three patients with ambiguous external genitalia and 45,X/46,XYnf karyotype is reported. The different clinical and cytogenetic aspects of this entity are described and discussed.     

Directed excision of a transgene from the plant genome

Summary The effectiveness of loxP-Cre directed excision of a transgene was examined using phenotypic and molecular analyses. Two methods of combining the elements of this system, re-transformation and cross pollination, were found to produce different degrees of excision in the resulting plants. Two linked traits, -glucuronidase (GUS) and a gene encoding sulfonylurea-resistant acetolactate synthase (ALS^r), were integrated into the genome of tobacco and Arabidopsis. The ALS^r gene, bounded by loxP sites, was used as the selectable marker for transformation. The directed loss of the ALS^T gene through Cre-mediated excision was demonstrated by the loss of resistance to sulfonylurea herbicides and by Southern blot analysis. The -glucuronidase gene remained active. The excision efficiency varied in F₁ progeny of different lox and Cre parents and was correlated with the Cre parent. Many of the lox × Cre F₁ progeny were chimeric and some F₂ progeny retained resistance to sulfonylureas. Re-transformation of lox/ALS/lox/GUS tobacco plants with cre led to much higher efficiency of excision. Lines of tobacco transformants carrying the GUS gene but producing only sulfonylurea-sensitive progeny were obtained using both approaches for introducing cre. Similarly, Arabidopsis lines with GUS activity but no sulfonylurea resistance were generated using cross pollinations.     

RAPD isolation of a Y chromosome specific ORF in a dioecious plant, Silene latifolia.

Silene latifolia is a dioecious plant and has heteromorphic sex chromosomes: the X and Y chromosomes. The Y chromosome is the largest, and its genetic control seems to be most strict among dioecious plants. To identify the putative sex-determination elements on the Y chromosome, random amplified polymorphic DNA (RAPD) analysis was used to screen for Y chromosome specific DNA fragments, and 31 clones were successfully produced. Genomic Southern hybridization and FISH (fluorescence in situ hybridization) analyses revealed that one of the clones, #2-2, is a Y chromosome specific fragment that has a single copy on the Y chromosome. Sequence tagged site (STS)-PCR analysis also succeeded in amplifying one fragment in males and no fragments in females. Cloning and sequencing of the #2-2 flanking region using inverse PCR revealed an open reading frame (ORF) corresponding to 285 amino acids in length (ORF285), but no expression of the ORF285 gene was identified. ORF285 may be a clue to the origin of dioecy.     

Y chromosome length variation and its significance in the horse

The results of Y chromosome measurements in 31 horses are presented. The Y chromosome was identified using G-, R-, and C-banding techniques. From G-banded metaphase spreads, total X and Y chromosome and separate proximal (P) and distal (D) Y-band measurements were made. Within this group, the Y/X ratio (%) for each animal varied from 18.93 to 43.95, with an overall mean of 34.85 and a coefficient of variation (CV) of 16.12. The overall mean P/X ratio (%) was 23.57 with a CV of 20.57, compared with an overall mean D/X ratio (%) of 11.26 with a CV of 15.18. The group studied included 27 Thoroughbred and 4-non-Thoroughbred animals, of which 20 were clinically normal controls and 11 presented with various clinical abnormalities. By comparison with data from other species, the possible breed association and clinical significance of the observed heteromorphism for the Y chromosome in this species is discussed.     

Origin and structure of a satellited Y chromosome

A chromosome Yqs was detected in a normal male. The origin and structure of this chromosome was investigated by means of different techniques: CBG, Ag-NOR, QFQ, THA, DA/DAPI and Distamycin A. The conclusion was reached that the Yqs chromosome was actually a Yq/15p translocation, where the Y chromosome had lost completely the Yq12 band. These findings suggested that the absence of the heterochromatic portion of the Y chromosome does not determine infertility.     

Characterization and evolution of a single-copy sequence from the human Y chromosome.

To study the evolution and organization of DNA from the human Y chromosome, we constructed a recombinant library of human Y DNA by using a somatic cell hybrid in which the only cytologically detectable human chromosome is the Y. One recombinant (4B2) contained a 3.3-kilobase EcoRI single-copy fragment which was localized to the proximal portion of the Y long arm. Sequences homologous to this human DNA are present in male gorilla, chimpanzee, and orangutan DNAs but not in female ape DNAs. Under stringent hybridization conditions, the homologous sequence is either a single-copy or a low-order repeat in humans and in the apes. With relaxed hybridization conditions, this human Y probe detected several homologous DNA fragments which are all derived from the Y in that they occur in male DNAs from humans and the apes but not in female DNAs. In contrast, this probe hybridized to highly repeated sequences in both male and female DNAs from old world monkeys. Thus, sequences homologous to this probe underwent a change in copy number and chromosomal distribution during primate evolution.     

Surnames and the Y chromosome

A randomly ascertained sample of males with the surname "Sykes" was typed with four Y-chromosome microsatellites. Almost half the sample shared the same Y-chromosome haplotype, which has not been observed in control samples either from the same geographic region or from the United Kingdom as a whole. This points to a single surname founder for extant Sykes males, even though written sources had predicted multiple origins. The distribution of other Sykes Y-chromosome haplotypes were not significantly different from those in controls and may be accounted for by the historical accumulation of nonpaternity during the past 700 years, in which case the average rate estimate is 1.3%/generation. If this pattern is reproduced with other surnames, it may have important forensic and genealogical applications.