Development Of Multiplex Pira-Pcr In Detecting Variants Associated With Blue Eggshell In Duck

Abstract

Blue eggshells in ducks have long intrigued researchers and farmers due to the economic benefits of the egg market, especially in Asia. Two G-to-A transitions in the cis-regulatory region of the ABCG2 gene were proposed to be responsible for the elevated expression of the ATP-binding cassette superfamily G member 2 (ABCG2) protein, which transports biliverdin and eventually leads to blue pigmentation of eggshells. However, no fast and precise methods have been developed to identify duck individuals with genetic variants causing blue eggshells. In this research, conventional and multiplex primer-introduced restriction analysis PCR (PIRA-PCR) were utilized to genotype the blue-eggshell-conferring nucleotide variants by creating synthetic restriction cut sites for restriction enzymes. The results unveiled two genotypes, GA/GA and GG/GA, with two previously discovered haplotypes, G-G and A-A, and a novel haplotype, G-A. The Sanger sequencing confirmed the effectiveness of multiplex PIRA-PCR, making it a reliable, accurate, and affordable genotyping assay