Molecular genotyping of chicken (Gallus gallus L.) feathering genes in connection with separation by sex

Traditional breeding is time and material consuming. Modern laboratory techniques significantly speed up and reduce the costs for breeding animals with the desired properties. A test based on the quantitative real-time PCR (qPCR) technology was developed to distinguish between homozygous and heterozygous state of the genes from alleles K and k which are responsible for the rate of wing feather growth in day-old chicks. The use of quantitative real-time PCR for the analysis of genotypes is aimed at the discrimination between one and two copies of the target gene in a genome. To obtain reliable results, certain rules must be followed when conducting the assay: the efficiency of the PCR should be close to the maximum; it is possible to obtain a significant number of false results without the appropriate statistical analysis. A new assay algorithm was proposed to overcome the limitations of qPCR: all samples are subjected to two successive independent analyses in parallel with the reference samples of both genotypes; if the two runs produce divergent results then the assay is repeated and the previous results are discarded. This approach allows to reduce assay error probability down to zero. The new system consists of three (instead of four) primers for amplification of two genes and two probes, allowing efficient analysis of various allele K genotypes. Quantitative real-time PCR data analysis was performed by ΔΔCt method using the statistics software package SPSS for ROC analysis. Using the method developed, the percentage of KK, Kk and kk genotypes was determined in 145 cocks of original lines B5, B6, B7 and B9 of domestic meat chicken of cross Smena 8. It was shown that 19 cocks of line B5 and 15 cocks of line B6 had kk genotype. From the 46 cocks of line B7, none had kk genotype, 17 cocks (37 %) had Kk genotype, and 29 cocks (63 %) had KK genotype. From the 65 cocks of line B9, none had kk genotype, 17 cocks (26 %) had Kk genotype, and 48 cocks (74 %) had KK genotype. Analyzed fragments were sequenced to exclude the effects of possible nucleotide sequence variability on the assay. The sequences did not contain any nucleotide substitutions in the sites of the primers and probes annealing. The data obtained will accelerate selection of new domestic meat chicken breeds with possibility of sexing based on feather length in day-old chicken. Further breeding work involves the assessment of the offspring using traditional and molecular genetic methods.