Cloning and identification of a female-specific DNA marker in Paramisgurnus dabryanus

Abstract

Paramisgurnus dabryanus (Cypriniformes; Cobitidae), has been an emerging aquaculture species in China since the 1990s. In this study, random amplified polymorphic DNA fingerprinting with 220 primers was used to identify a sex-specific DNA marker in pooled DNA and individual DNA samples from male and female P. dabryanus. One primer, S2115, produced a novel sex-specific DNA fragment found only in tested females. This female-specific fragment was 917bp with 36% GC content, and was named Pdff1. To further validate the authenticity of this female-specific marker for sexing, two PCR primers (pdff1-F and -R) were designed according to the cloned female-specific sequence. Amplification showed bands specific for females. Dot blot and Southern blot hybridization experiments both displayed female specificity using this marker as the probe. Two other P. dabryanus populations were tested by dot blot hybridization with the Pdff1 probe. The hybridization signals were seen in 33 or 43% of males in addition to all females in the Jinan and Xichuan populations, respectively. We propose to use this sex-specific marker to rapidly and specifically identify the gender of P. dabryanus from the ancient Yellow River Wetland in Yanjin, Henan Province. Our results could assist in cloning sex-specific chromosomal regions.