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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. 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. [ABSTRACT FROM AUTHOR]

Feather colors of chickens are not only characteristics of breeds but also as phenotypic markers in chicken breeding. Pure-bred Rhode Island Red (RIR) chicks have a stripe pattern and a non-stripe pattern on the back. The stripe pattern of RIR is generally shown as four longitudinal black stripes on the back and is more likely to appear in females. In this study, we performed a genome-wide association study (GWAS) to identify candidate genes controlling the stripe pattern of RIR chicks, and then, based on physical location and biological functions, quantitative RT-PCR analysis was used to validate the differential expression of candidate genes between stripe pattern and non-stripe pattern back skin tissue. The GWAS showed that a major signal contains 768 significant single nucleotide polymorphisms (SNPs) and 87 significant small insertions-deletions (INDELs) spanning 41.78 to 43.05 Mb (~1.27 Mb) on GGA1, corresponding to 16 genes associated with stripe pattern phenotype. Among these 16 genes, KITLG and TMTC3 could be considered candidate genes as they showed different expressions between back skin tissues of stripe pattern and non-stripe pattern chicks in value (p = 0.062) and the significant level (p < 0.05), respectively. This study provided novel insight into the mechanisms underlying feather pigmentation and stripe formation in RIR chicks. [ABSTRACT FROM AUTHOR]