The location of Z- and W-linked marker genes and sequence on the homomorphic sex chromosomes of the ostrich and the emu

Abstract

Perhaps the most striking fact about early Cenozoic avian history some 70 million years ago was the rapid radiation of large, flightless, ground-living birds. It has been suggested that, for a time, there was active competition between these large terrestrial birds and the early mammals. Probably reflecting the above noted early start of Ratitae of the infraclass Eoaves, the presumptive sex chromosomes of their present day survivors, such as the emu and the ostrich, largely remained homomorphic. The signs of genetic differentiation between their still-homomorphic Z and W chromosomes were tested by using two marker genes (Z-linked ZOV3 and the gene for the iron-responsive element-binding protein) and one marker sequence of a part of a presumptive pseudogene (W-linked EE0.6 of the chicken). Their homologues, maintaining 71-92% identities to the chicken counterparts, were found in both the emu (Dromaius novaehollandiae) and the ostrich (Struthio camelus). Their locations were visualized on chromosome preparations by fluorescence in situ hybridization. In the case of the emu, these three marker sequences were localized on both members of the fifth pair of a female, thus revealing no sign yet of genetic differentiation between the Z and the W. The finding was the same with regard to both members of the fourth pair of male ostriches. In the female ostrich, however, the sequence of the gene for the iron-responsive element-binding protein was missing from one of the pairs, thus revealing the differentiation by a small deletion of the W from the Z.

Figure 1

Localization of IREBP, ZOV3, and EE0.6 sequences on a pair of chromosomes in metaphase sets of the emu and the ostrich. Signals of FISH shown by fluorescein isothiocyanate (white dots) or tetramethylrhodamine isothiocyanate (red dots) fluorescence indicate hybridization of the following probes: (A) DIG-pEGIREBP (arrowhead) and biotinylated pEGZOV3 (triangle) to the female emu set; (B) biotinylated pOGZOV3 (triangle) and DIG-pOGIREBP (arrowhead) to the male ostrich set; (C) as in B, but to the female ostrich set; (D) biotinylated pEGIREBP (arrowhead) and DIG-pEGEE0.6 (arrow) to the female emu set; (E) biotinylated pOGIREBP (arrowhead) and DIG-pOGEE0.6 (arrow) to the male ostrich set; and (F) as in E but to the female ostrich set. (Bars = 10 μm.)

Figure 2

Identification of the pair of homologous chromosomes on which ZOV3, IREBP, and EE0.6 sequences are colocalized and partial morphological differentiation of the W chromosome in the ostrich. Chromosome pairs 1–6 from in situ-hybridized metaphase sets of the female emu (A) and the male ostrich (B) are shown. Signals of FISH are colocalized on chromosome 5 in the emu and chromosome 4 in the ostrich. Fluorescence signals were produced with the following probes: biotinylated pEGIREBP (arrowhead) and DIG-pEGEE0.6 (arrow) in A and DIG-pOGIREBP (arrowhead) and biotinylated pOGZOV3 (triangle) in B. (C) Pairs of chromosome 4 of the ostrich were taken from in situ-hybridized metaphase sets of the male, the female, and the individuals (nos. 7 and 8) of unknown gender. Fluorescence signals shown were produced with biotinylated pOGZOV3 (triangle) and DIG-labeled pOGIREBP (arrowhead). Partial differentiation of the W chromosome in the female and the no. 8 ostriches is noted by shortening of the arm from which the IREBP gene is missing.

Figure 3

Southern blot hybridization suggesting the absence of the IREBP gene on one of the homologous chromosomes in the female ostrich. The BglII-digested genomic DNA samples (5 μg/lane) from the male and female ostriches and from the two ostriches (nos. 7 and 8) of unknown gender as indicated were subjected to hybridization with the following ³²P-labeled probes: (A) a 2.4-kb XbaI fragment of pOGEE0.6; (B) a 2.7-kb EcoRI–BglII fragment of pOGIREBP; and (C) a 3.5-kb BamHI fragment of pOGZOV3. The same blot was hybridized and rehybridized in this order. Relative intensities of hybridization signals are indicated.