O.V. Romanenkova, E.A. Gladyr, O.V. Kostjunina, N.A. Zinovieva
L.K. Ernst Institute for Animal Husbandry, Dubrovitzy, 60, Podolskiy r-n, Moskovskaya obl., 142132, Russian Federation
Summary. Recessive genetic disorders, which are associated with embryonic mortality, are seen as one of the significant causes of reduced cow fertility. The aim of this work was to develop a molecular genetic test system for screening of nonsense mutation with substitution of C by T in APAF1 gene coding for apoptotic protease activating factor 1 and causing substitution of Q by X in 579 position of amino acid sequence, which is associated with fertility haplotype HH1, and to apply it for screening of Russian Holstein cattle for HH1. Using direct DNA sequence analysis by parasequencing of the region of localization of causative mutation, we created the set of reference samples with known genotypes for APAF1 (n = 80), including 66 samples with QQ genotype (non-carrier of HH1) and 14 samples with QX genotype (HH1 carrier). The proposed test system is based on PCR amplification of APAF1 fragment containing mutation with the subsequent hydrolysis of PCR products by restriction enzyme BstC8I. To exclude the non-specific restriction site for BstC8I which is located 10 bp down-stream of mutation, we incorporated the nucleotide substitution in reverse primer. Analysis of reference samples using the developed test system showed full concordance of results of genotyping. Thirty-nine carriers of HH1 including 23 sires and 16 cows were identified among 593 tested sires and 270 cows, which corresponds to the carriers’ frequencies of 3.9 and 5.9%, respectively. To control the spread of HH1 carriers among Russian breeding population of Holstein cattle and cattle carrying the Holstein blood we recommend the application of developed test system for diagnostics of APAF1 polymorphism.
Keywords: cattle, recessive genetic disorders, fertility haplotypes, DNA diagnostics.
Acknowledgments: The studies were performed under financial support of the Russian Ministry of Education and Science (unique project identification number RFMEFI60414X0062).
Author Details: O.V. Romanenkova, post-graduate student; E.A. Gladyr, Сand. Sc. (Biol.), head of laboratory; O.V. Kostjunina, Сand. Sc. (Biol.), head of division; N.A. Zinovieva, D. Sc. (Biol.), member of the RAS, director (e-mail: Этот адрес электронной почты защищён от спам-ботов. У вас должен быть включен JavaScript для просмотра.).
For citation: Romanenkova O.V., Gladyr E.A., Kostjunina O.V., Zinovieva N.A. Screening of Cattle in Russia for the Presence of Mutation in APAF1 Gene, which Is Associated with Fertility Haplotype HH1. Dostizheniya nauki i tekhniki APK. 2016. V.30. No 2. Pp. 94-97 (In Russ.).