Antonio Bernardo Carvalho

Identification of five new genes on the Y chromosome of Drosophila melanogaster

The heterochromatic state of the Drosophila Y chromosome has made the cloning and identification of Y-linked genes a challenging process. Here, we report application of a procedure to identify Y-linked gene fragments from the unmapped residue of the whole genome sequencing effort. Previously identified Y-linked genes appear in sequenced scaffolds as individual exons, apparently because many introns have become heterochromatic, growing to enormous size and becoming virtually unclonable. A TBLASTN search using all known proteins as query sequences, tested against a blastable database of the unmapped fragments, produced a number of matches consistent with this scenario. Reverse transcription–PCR and genetic methods were used to confirm those that are expressed, Y-linked genes. The five genes reported here include three protein phosphatases (Pp1-Y1, Pp1-Y2, and PPr-Y), an occludin-related gene (ORY), and a coiled-coils gene (CCY). This brings the total to nine protein-coding genes identified on the Drosophila Y chromosome. ORY and CCY may correspond, respectively, to the fertility factors ks-1 and ks-2, whereas the three protein phosphatases represent novel genes. There remains a strong functional coherence to male function among the genes on the Drosophila Y chromosome.

Low conservation of gene content in the Drosophila Y chromosome

Chromosomal organization is sufficiently evolutionarily stable that large syntenic blocks of genes can be recognized even between species as distantly related as mammals and puffer fish (450 million years (Myr) of divergence). In Diptera, the gene content of the X chromosome and the autosomes is well conserved: in Drosophila more than 95% of the genes have remained on the same chromosome arm in the 12 sequenced species (63 Myr of divergence, traversing 400 Myr of evolution), and the same linkage groups are clearly recognizable in mosquito genomes (260 Myr of divergence). Here we investigate the conservation of Y-linked gene content among the 12 sequenced Drosophila species. We found that only a quarter of the Drosophila melanogaster Y-linked genes (3 out of 12) are Y-linked in all sequenced species, and that most of them (7 out of 12) were acquired less than 63 Myr ago. Hence, whereas the organization of other Drosophila chromosomes traces back to the common ancestor with mosquitoes, the gene content of the D. melanogaster Y chromosome is much younger. Gene losses are known to have an important role in the evolution of Y chromosomes, and we indeed found two such cases. However, the rate of gene gain in the Drosophila Y chromosomes investigated is 10.9 times higher than the rate of gene loss (95% confidence interval: 2.3–52.5), indicating a clear tendency of the Y chromosomes to increase in gene content. In contrast with the mammalian Y chromosome, gene gains have a prominent role in the evolution of the Drosophila Y chromosome.

Intron size and natural selection

Intron sizes vary widely among different genes and among homologous genes of different species. The distribution of intron sizes may be maintained in a steady state, reflecting the processes of insertion and deletion of gene sequences, or it may be that the distribution is constrained by natural selection. If intron size is governed by natural selection, there should be a statistical association between this size and the rate of recombination per map unit of the genome, assuming that natural selection is less effective in genomic regions of low recombination. Here we show that larger introns of Drosophila melanogaster occur preferentially in regions of low recombination, which is consistent with large introns having a deleterious effect. The association is significant (P=40.001, linear regression), despite the fact that no effort was made to stratify the data by other factors that affect intron size, such as the size of the associated coding region or the presence of regulatory sequences inside the intron.

Genetic recombination: Intron size and natural selection

Intron sizes vary widely among different genes and among homologous genes of different species. The distribution of intron sizes may be maintained in a steady state, reflecting the processes of insertion and deletion of gene sequences, or it may be that the distribution is constrained by natural selection. If intron size is governed by natural selection, there should be a statistical association between this size and the rate of recombination per map unit of the genome, assuming that natural selection is less effective in genomic regions of low recombination. Here we show that larger introns of Drosophila melanogaster occur preferentially in regions of low recombination, which is consistent with large introns having a deleterious effect. The association is significant (P=40.001, linear regression), despite the fact that no effort was made to stratify the data by other factors that affect intron size, such as the size of the associated coding region or the presence of regulatory sequences inside the intron.

Efficient identification of Y chromosome sequences in the human and Drosophila genomes

Notwithstanding their biological importance, Y chromosomes remain poorly known in most species. A major obstacle to their study is the identification of Y chromosome sequences; due to its high content of repetitive DNA, in most genome projects, the Y chromosome sequence is fragmented into a large number of small, unmapped scaffolds. Identification of Y-linked genes among these fragments has yielded important insights about the origin and evolution of Y chromosomes, but the process is labor intensive, restricting studies to a small number of species. Apart from these fragmentary assemblies, in a few mammalian species, the euchromatic sequence of the Y is essentially complete, owing to painstaking BAC mapping and sequencing. Here we use female short-read sequencing and k-mer comparison to identify Y-linked sequences in two very different genomes, Drosophila virilis and human. Using this method, essentially all D. virilis scaffolds were unambiguously classified as Y-linked or not Y-linked. We found 800 new scaffolds (totaling 8.5 Mbp), and four new genes in the Y chromosome of D. virilis, including JYalpha, a gene involved in hybrid male sterility. Our results also strongly support the preponderance of gene gains over gene losses in the evolution of the Drosophila Y. In the intensively studied human genome, used here as a positive control, we recovered all previously known genes or gene families, plus a small amount (283 kb) of new, unfinished sequence. Hence, this method works in large and complex genomes and can be applied to any species with sex chromosomes.

Sex-ratio in Drosophila mediopunctata

The occurrence of sex-ratio in Drosophila mediopunctata is described. The sex-ratio trait, affected males producing progenies with a large excess of females, is known also in eight other Drosophila species. It has X-linked inheritance, being apparently always associated with particular X chromosome inversions. The expression of the sex-ratio trait in D. mediopunctata is very variable.

Autosomal suppressors of sex-ratio in Drosophila mediopunctata

The sex-ratio trait has been described as the production of progenies with excess of females due to X-linked meiotic drive in the parental males. This trait has a variable expression in Drosophila mediopunctata. We describe here the existence and chromosomal localization of autosomal suppressors of sex-ratio in this species. There are at least four such genes (one on each major autosome) and the strongest effect is localized on chromosome IV. These genes possibly result from the operation of ‘Fishers Principle’; a mechanism of Natural Selection leading to a 1:1 sex ratio.

Y-linked suppressors of the sex-ratio trait in Drosophila mediopunctata

X-linked meiotic drive causing female-biased progenies is known to occur in nine Drosophila species and is called ‘sex-ratio’. In D. mediopunctata this trait is associated with the X:21 chromosome inversion and has variable expression. We describe here a powerful Y-linked suppressor system of sex-ratio expression in this species. There are two types of Y chromosomes (suppressor and nonsuppressor) and two types of X:21 chromosomes (suppressible and unsuppressible). Sex-ratio expression is suppressed in males with the 21suppressibe/Ysuppressor genotype, whereas the remaining three genotypes produce female-biased progenies.X-linked meiotic drive causing female-biased progenies is known to occur in nine Drosophila species and is called ‘sex-ratio’. In D. mediopunctata this trait is associated with the X:21 chromosome inversion and has variable expression. We describe here a powerful Y-linked suppressor system of sex-ratio expression in this species. There are two types of Y chromosomes (suppressor and nonsuppressor) and two types of X:21 chromosomes (suppressible and unsuppressible). Sex-ratio expression is suppressed in males with the 21suppressibe/Ysuppressor genotype, whereas the remaining three genotypes produce female-biased progenies.

Birth of a new gene on the Y chromosome of Drosophila melanogaster

Significance Mammalian Y chromosomes are believed to evolve mainly through gene inactivation and loss. Drosophila Y chromosomes seem to not obey this rule, as gene gains are the dominating force in their evolution. Here we describe flagrante delicto Y (FDY), a very young gene that shows how Y-linked genes were acquired. FDY originated 2 million years ago from a duplication of a contiguous autosomal segment of 11 kb containing five genes that inserted into the Y chromosome. Four of these autosome-to-Y gene copies became inactivated (“pseudogenes”), lost part of their sequences, and most likely will disappear in the next few million years. FDY, originally a female-biased gene, acquired testis expression and remained functional. Contrary to the pattern seen in mammalian sex chromosomes, where most Y-linked genes have X-linked homologs, the Drosophila X and Y chromosomes appear to be unrelated. Most of the Y-linked genes have autosomal paralogs, so autosome-to-Y transposition must be the main source of Drosophila Y-linked genes. Here we show how these genes were acquired. We found a previously unidentified gene (flagrante delicto Y, FDY) that originated from a recent duplication of the autosomal gene vig2 to the Y chromosome of Drosophila melanogaster. Four contiguous genes were duplicated along with vig2, but they became pseudogenes through the accumulation of deletions and transposable element insertions, whereas FDY remained functional, acquired testis-specific expression, and now accounts for ∼20% of the vig2-like mRNA in testis. FDY is absent in the closest relatives of D. melanogaster, and DNA sequence divergence indicates that the duplication to the Y chromosome occurred ∼2 million years ago. Thus, FDY provides a snapshot of the early stages of the establishment of a Y-linked gene and demonstrates how the Drosophila Y has been accumulating autosomal genes.

Long-Read Single Molecule Sequencing to Resolve Tandem Gene Copies: The Mst77Y Region on the Drosophila melanogaster Y Chromosome.

The autosomal gene Mst77F of Drosophila melanogaster is essential for male fertility. In 2010, Krsticevic et al. (Genetics 184: 295−307) found 18 Y-linked copies of Mst77F (“Mst77Y”), which collectively account for 20% of the functional Mst77F-like mRNA. The Mst77Y genes were severely misassembled in the then-available genome assembly and were identified by cloning and sequencing polymerase chain reaction products. The genomic structure of the Mst77Y region and the possible existence of additional copies remained unknown. The recent publication of two long-read assemblies of D. melanogaster prompted us to reinvestigate this challenging region of the Y chromosome. We found that the Illumina Synthetic Long Reads assembly failed in the Mst77Y region, most likely because of its tandem duplication structure. The PacBio MHAP assembly of the Mst77Y region seems to be very accurate, as revealed by comparisons with the previously found Mst77Y genes, a bacterial artificial chromosome sequence, and Illumina reads of the same strain. We found that the Mst77Y region spans 96 kb and originated from a 3.4-kb transposition from chromosome 3L to the Y chromosome, followed by tandem duplications inside the Y chromosome and invasion of transposable elements, which account for 48% of its length. Twelve of the 18 Mst77Y genes found in 2010 were confirmed in the PacBio assembly, the remaining six being polymerase chain reaction−induced artifacts. There are several identical copies of some Mst77Y genes, coincidentally bringing the total copy number to 18. Besides providing a detailed picture of the Mst77Y region, our results highlight the utility of PacBio technology in assembling difficult genomic regions such as tandemly repeated genes.