Development of innovative marker detection methods for high-fertility ducks (Anas plastyrhynchos)

We previously analyzed the genome-wide gene expression at the transcription level in pre-hierarchical ovarian follicles (approximate 5 mm in diameter) between two groups of ducks representing high and low fertility. Orthodenticle homeobox 2 (otx2) was identified with significantly differential expression in the high-fertility group versus the low-fertility group. To identify the relationships between genotypes and phenotypes, we recorded the reproductive performance in advance, including fertility, hatchability, and fertile period of female ducks. To ensure coverage of the entire duration of the fertile period, we extended the egg collection period after artificial insemination. Naturally, sperm cannot survive after a certain period of time in the female reproductive tract (sperm is not immortal); therefore, lower average values for fertility were observed in this study than that observed after a normal egg collection period, i.e., the lower average values of fertility (18 days after artificial insemination), were not due to the effect of otx2. The otx2 genomic sequence of Tsaiya ducks was firstly amplified with a primer pair of i3F and i3R for polymerase chain reaction based on Pekin duck sequence and a resultant 444-base pair fragment was obtained for DNA sequencing. Using multiple sequence alignment, new single-nucleotide polymorphisms g.366T > C and g.182G > T were discovered in the otx2 gene. With respect to g.366T > C, ducks were classified into CC, CT, and TT genotypes. For g.182G > T, three genotypes (GG, GT, and TT) were identified. Ducks were genotyped using novel specific primers and probes to rapidly screen their single-nucleotide polymorphisms. The results indicated that ducks with the CC genotype of g.366T > C exhibited the highest fertility among the CC, CT, and TT genotypes (p < 0.05). No significant difference was found in the fertile period and hatchability among three genotypes of g.366T > C. Moreover, no association was found between g.182G > T genotypes and the three reproductive phenotypes examined in this study. Collectively, the otx2 g.366T > C genotype is associated with duck females, and can be used as a marker for farming a flock of ducks with high fertility, as well as for genetic selection of breeders.