A new primer-pair for sex identification of larks and wagtails

Chromobox-helicase DNA-binding gene (CHD1) is the most reliable gene for sex identification in birds. However, the CHD1 fragments of some species are difficult to amplify. In this study, we designed a new primer set (IntP2/IntP8) that targets a conserved region of CHD1 gene. Firstly, we tested this protocol in Oriental skylark (Alauda gulgula). PCR amplification produced a single band (259 bp) for males and two bands (259 and 297 bp) for females. We then successfully conducted sex identification in other bird species including Tibetan lark (Melanocorypha maxima), Horned lark (Eremophila alpestris), Mongolian skylark (Melanocorypha mongolica), Asian short-toed lark (Calandrella cheleensis), Humes short-toed lark (Calandrella acutirostris), Crested lark (Galerida cristata), Yellow-headed wagtail (Motacilla citreola), White wagtail (Motacilla alba) and Ground tit (Pseudopodoces humilis Hume). Collectively, these findings support that IntP2/IntP8 primer set can be used to accurately determine sex identity in birds.