Itoh Y, Suzuki M, Ogawa A, Munechika I, Murata K, Mizuno S
Laboratory of Molecular Biology, Department of Molecular and Cell Biology, Graduate School of Agricultural Science, Tohoku University, 1-Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai, Japan.
J Hered. 2001 Jul-Aug;92(4):315-21. doi: 10.1093/jhered/92.4.315.
A 0.6 kb EcoRI fragment (EE0.6), cloned from the W chromosome of chickens, is a nonrepetitive sequence and contains an exonlike sequence, ET15, which is likely a part of a pseudogene. The EE0.6 sequence is conserved in all species of birds examined both in Carinatae and Ratitae. A counterpart sequence of EE0.6 is present on the Z chromosome. The extent of diversity between the W- and Z-linked sequences are variable among species. The W- and Z-linked EE0.6 sequences, cloned from 12 different species, were compared and four forward and three reverse primers were selected to amplify parts of the EE0.6 sequence by polymerase chain reaction (PCR). By choosing a suitable combination of primers for EE0.6 and a set of primers for a Z/W-common sequence, as an internal control, the sex of 36 species belonging to 16 different orders of Carinatae could be determined clearly by PCR. The sex of two other species representing different orders could be determined by Southern blot hybridization using ET15 as a probe. For the two Ratitae species, emu and ostrich, EE0.6 sequences on W and Z chromosomes could not be distinguished either by PCR or Southern blotting.
从鸡的W染色体上克隆的一个0.6 kb的EcoRI片段（EE0.6）是一个非重复序列，包含一个外显子样序列ET15，它可能是一个假基因的一部分。EE0.6序列在所检测的所有鸡形目和鸵形目鸟类物种中都是保守的。EE0.6在Z染色体上有对应的序列。W染色体和Z染色体上的序列之间的差异程度在不同物种中有所不同。对从12个不同物种中克隆的W染色体和Z染色体上的EE0.6序列进行了比较，并选择了4条正向引物和3条反向引物，通过聚合酶链反应（PCR）扩增EE0.6序列的部分片段。通过选择适合EE0.6的引物组合以及一组用于Z/W共同序列的引物作为内部对照，利用PCR可以明确确定鸡形目16个不同目36个物种的性别。另外两个代表不同目的物种的性别可以通过以ET15为探针的Southern印迹杂交来确定。对于鸵形目的鸸鹋和鸵鸟这两个物种，无论是通过PCR还是Southern印迹法都无法区分W染色体和Z染色体上的EE0.6序列。
