Regulatory function and effective variation identification of egg-laying related candidate gene ANK2 in chickens

Though numerous egg-laying-related candidate genes and loci have been uncovered, most studies merely establish phenotypic correlations, lacking functional verification and variant identification, which limits molecular breeding application. This study systematically investigated the regulatory function and effective functional variations of the laying-related candidate gene Ankyrin 2 (ANK2) in chickens. First, through population genetic analysis and multi-type GWAS analysis based on ANK2 genomic region, we confirmed that ANK2 underwent strong artificial selection during chicken domestication and laying-oriented breeding, and multiple SNPs of this gene were significantly associated with egg number and clutch traits, demonstrating the genetic basis of ANK2 as a key candidate gene for chicken egg-laying traits. Expression characteristic analysis in hypothalamic-pituitary-ovarian axis showed that ANK2 was only differentially expressed in ovaries of the high- and low-laying hens, with its ovarian expression positively correlated with egg-laying traits, and was dynamically expressed during follicular development. Further, knockdown of ANK2 altered the transcriptome of chicken ovarian granulosa cells, suppressed steroid synthesis-related genes and progesterone secretion, and inhibited granulosa cell proliferation and differentiation. Finally, three candidate effective variations of ANK2 were screened from two indigenous chicken breeds, and one SNP chr4:56869062C>T was verified to help account for the differences in egg-laying phenotypes among chicken populations by regulating ANK2 expression, with its C allele enriched in high-laying breeds. This research provides functional gene and molecular marker resources for chicken laying performance molecular breeding.