Comparative Genomic Hybridization (CGH) reveals a neo-X chromosome and biased gene movement in stalk-eyed flies (genus Teleopsis)

Abstract

Chromosomal location has a significant effect on the evolutionary dynamics of genes involved in sexual dimorphism, impacting both the pattern of sex-specific gene expression and the rate of duplication and protein evolution for these genes. For nearly all non-model organisms, however, knowledge of chromosomal gene content is minimal and difficult to obtain on a genomic scale. In this study, we utilized Comparative Genomic Hybridization (CGH), using probes designed from EST sequence, to identify genes located on the X chromosome of four species in the stalk-eyed fly genus Teleopsis. Analysis of log(2) ratio values of female-to-male hybridization intensities from the CGH microarrays for over 3,400 genes reveals a strongly bimodal distribution that clearly differentiates autosomal from X-linked genes for all four species. Genotyping of 33 and linkage mapping of 28 of these genes in Teleopsis dalmanni indicate the CGH results correctly identified chromosomal location in all cases. Syntenic comparison with Drosophila indicates that 90% of the X-linked genes in Teleopsis are homologous to genes located on chromosome 2L in Drosophila melanogaster, suggesting the formation of a nearly complete neo-X chromosome from Muller element B in the dipteran lineage leading to Teleopsis. Analysis of gene movement both relative to Drosophila and within Teleopsis indicates that gene movement is significantly associated with 1) rates of protein evolution, 2) the pattern of gene duplication, and 3) the evolution of eyespan sexual dimorphism. Overall, this study reveals that diopsids are a critical group for understanding the evolution of sex chromosomes within Diptera. In addition, we demonstrate that CGH is a useful technique for identifying chromosomal sex-linkage and should be applicable to other organisms with EST or partial genomic information.

Figure 1. Distribution of T. dalmanni CGH log₂ ratio values.

Male and female genomic DNA were hybridized to microarray slides containing probes designed from EST sequence data. A total of 3444 genes are represented. The large peak indicates autosomal genes while the smaller peak are genes on the X chromosome. Dark bars indicate the log₂ ratio distribution for all the genes, while the hatched bars provide the log₂ ratio distribution for the 132 genes that violate the syntenic relationship between the X chromosome in T. dalmanni and the 2L chromosome in D. melanogaster.

Figure 2. T. dalmanni chromosomal synteny with D. melanogaster.

The autosomal category includes 2891 T. dalmanni genes and the X chromosome comprises 533 T. dalmanni genes. Homology between D. melanogaster and T. dalmanni was assessed based on BlastX searches of EST sequence .

Figure 3. Chromosomal linkage map of 28 annotated EST genes in T. dalmanni.

Unlabeled hashmarks indicate positions of microsatellite markers. This map was created using genotypes of 639 flies (288 males, 351 females) from two F2 families obtained by crossing lines of flies selected for increased or decreased eye stalk length [cf. 40]. 23 markers were informative in both families and provided the framework for estimating linkage relationships for markers that were only informative in a single family. Color-coding indicates the chromosomal arm location in D. melanogaster. As indicated by CGH, four X-linked loci are found on 2L. Furthermore, one autosome appears to be a fusion of X+3L while the other autosome contains genes predominantly from 2R and 3R.

Figure 4. Distribution of CGH log₂ ratio values for three other Teleopsis species.

Male and female genomic DNA were hybridized to microarray slides containing probes designed from T. dalmanni EST sequence data. Approximately 3400 genes are represented in each histogram. The large peak indicates autosomal genes while the smaller peak are genes on the X chromosome. A) Teleopsis whitei. B) Teleopsis thaii. C) Teleopsis quinqueguttata.

Figure 5. Gene movement in Teleopsis.

Reconstruction of gene movement between autosomes and the X chromosome was polarized using chromosomal data from A. gambiae and D. melanogaster. The X chromosome in Teleopsis was assumed to be homologous to chromosome 3R in A. gambiae and 2L in D. melanogaster. The branches with hash marks represent those that have undergone increases in eye-stalk sexual dimorphism. The states for each species and the specific genes associated with each reconstruction (using the branch numbers as reference) are presented in Table S2.

Figure 6. Relationship between Teleopsis chromosomal location and the rate of protein evolution.

The rate of protein evolution in Teleopsis was measured as the branch length leading to T. dalmanni expressed as a percentage of the entire tree length based a phylogenetic analysis including A. gambiae and three Drosophila species . The sample sizes for the chromosomal locations are: A - 2034 genes, Onto A – 41 genes, X – 342 genes and Onto X – 23 genes.