Molecular identification, isolation and evolution of sex -chromosomal DNA sequence in cranes

A female-specific DNA fragment (CSL-W; Crane Sex Linked DNA on W chromosome) was isolated from whooping cranes (Grus americana). From the nucleotide sequence, PCR primers were identified which amplify a female specific fragment from all crane species and some non-crane species. A duplicated version of the segment was also identified in both sexes, and designated Crane Sex linked DNA Non-W chromosome (CSL-NW). The CSL primers do not amplify mammalian DNA, limiting the potential for human contamination. A test of the methodology was carried out on feathers from 18 captive cranes, resulting in 100% correct sex identification. The low cost, speed, simplicity and accuracy of the molecular methods, and storage, transport and non-invasive nature of collection of feather samples are major advantages of the method.;Since the CSL and the Chromo-helicase-DNA-binding protein (CHD) genes were located on both W and Z chromosomes (or on an autosome), a heteroduplex technique was used to detect difference between W-linked allele and its paralogous allele. The PCR products appeared as a single band when the PCR product was analyzed with 3% agarose or agrose-syner gel electrophoresis. After the PCR products were treated by heteroduplex procedures, a duplex band was formed in females. Since the males have only the Z chromosome, a single product was detected in males.;It has been suggested that evolution of DNA sequences is male driven in mammals because of a higher substitution rate in genes on the Y chromosome. In birds, evolution would be different since the Z chromosome occurs as two copies in the male, while female birds carry a W chromosome. A 283--285 bp CHD-Z intron and a 289--293 CSL-NW DNA fragment were sequenced from males of all species of cranes. The Z linked intron evolves very slowly and identical sequences were obtained from five species that diverged 1--3 million years ago. Relative substitution rate in the crane CHD-Z intron is similar to that of woodpecker nuclear beta fibrinogen intron. A phylogenetic based on DNA sequences from the CHD intron and the CSL sequence shows the three major lineages hypothesized within the crane family.