Identification of Duplication Genotypes of the Feathering Rate Gene in Chicken by a Multiplex PCR Following Electrophoresis and/or Sanger Sequencing

SIMPLE SUMMARY: Autosexing based on sex-linked phenotypes of late feathering (LF) and early feathering (EF) was widely used in poultry production. Previous studies found that tandem duplication on the Z chromosome is responsible for LF. In this study, based on tandem duplication, we developed a multiplex PCR test for genotyping through reaction optimization. The genotyping result for feathering rate with the multiplex PCR test was consistent with the sex and phenotype records, and the result of homozygote and heterozygote tests of LF males were verified by test cross and tallied with offspring’s phenotypes. The multiplex PCR test is stable and accurate for feathering rate genotyping, applicable to all LF chickens associated with tandem duplication, and has great significance for poultry breeding. ABSTRACT: Sex-linked phenotypes of late feathering (LF) and early feathering (EF) are controlled by a pair of alleles K and k(+). Autosexing based on the feathering rate is widely used in poultry production. It is reported that a tandem duplication of 176,324 base pairs linked to the K locus is responsible for LF expression and could be used as a molecular marker to detect LF chicken. So far, there is no genotyping method that can accurately and stably identify the LF homozygote and heterozygote in all chicken breeds. In the present study, a multiplex PCR test was developed to identify EF, LF homozygote, and heterozygote according to electrophoretic bands and the relative height of the peaks by Sanger sequencing. We tested 413 chickens of six native Chinese breeds with this method. The identification was consistent with the sex and phenotype records of the chickens. Band density analysis was performed, and the results supported our genotyping using the new assay. In order to further verify the accuracy of this test in distinguishing homozygote and heterozygote males, 152 LF males were mated with EF females, and the results of the offspring’s phenotypes were consistent with our expectations. Our results support tandem duplication as molecular markers of LF, and this new test is applicable to all LF chickens associated with tandem duplication.