Improved molecular assay for sex identification of the endangered crested Ibis (Nipponia nippon) based on the CHD1 gene and a sex-linked microsatellite locus.

Sex Identification of monomorphic birds, especially endangered avian species, is essential for ecological study and biodiversity conservation. In this study, two popular primer sets of 2550F/2718R and P2/P8, which were designed to amplify different fragments of chromodomain-helicase-DNA binding protein 1 (CHD1) genes mapped on both Z and W chromosomes in birds, were used to identify for the first time the sex of individuals of the endangered species crested ibis (Nipponia nippon) in a large number of samples. An improved primer set of 2467F/2530R was re-designed to be specific to crested ibis following their conserved sequences derived from the 2550F/2718R primers. PCR products of the new primers were conveniently visualized with two bands of 552 base pairs (bp) and 358 bp for females, but a single band of 552 bp for males in routine 1.8% agarose gel. Similarly, the P2/P8 primer set amplified two fragments of 398 bp and 381 bp from females but one fragment of 398 bp from males; however, a high resolution involving 10% Polyacrylamide gel had to be employed to resolve the 17 bp insertions/deletions (in/dels) present between the two amplicons in females. In addition, a microsatellite locus NnNF05 was validated to be sex-linked and shown to be effective in the sexing of crested ibis, supporting its utility in non-invasive sampling. This study provides a rapid, convenient, and reliable molecular assay for improving sex identification in the monomorphic and monogamous crested ibis, and thus facilitates the selection of breeding pairs in captive programs and reintroduction initiatives.