DNA analysis of myostatin, leptin and calpain 1 gene polymorphism in Russian cattle population of Aberdeen Angus breed

Beef meat is characterized by high nutritional value and unique amino acid composition (V.S. Kolodyaznaya et al., 2011; D. Pighin et al., 2016). Specialized beef cattle breeds in particular Aberdeen Angus ensures good acclimatization ability and high productivity both in Russia and broad (R. Toušová et al., 2015; V.M. Gabidulin et al., 2018; A.I. Otarov et al., 2021). Modern strategies of increasing efficiency of meat cattle farming include animal genotyping for genetic determinants of high productivity and targeted selection (V.F. Fedorenko et al., 2018; S.A. Terry et al., 2020). This paper is the first to report the development of a PCR-RFLP-based test for detection of Arg4Cys LEP and CAPN1 _316 allele polymorphisms. This test was used for genotyping of the Aberdeen-Angus cattle population. The frequencies of the different polymorphism genotypes are evaluated and the influence of the LEP and CAPN1 polymorphisms on animal fattening qualities. The purpose of the study was to develop the test systems for revealing the leptin LEP gene c.466 C→T polymorphism and calpain 1 CAPN1 gene polymorphism rs17872000, to genotype Aberdeen Angus cattle population for these genes and the myostatin MSTN gene, and to investigate the links between different allele variants and fattening qualities of animals. Bulls ( n = 64) and heifers ( n = 81) of the population of Aberdeen-Angus young cattle ( Bos taurus ) (OOO Happy Farm, Medynsky District, Kaluga Province) born from March 2020 until May 2021 were selected for the study. Weaning of calves from mothers was carried out at the age of 6-9 months, depending on the development of a particular calf. The live weight was determined at the age of 6, 8, 12 and 15 months. Blood samples were collected for genotyping. DNA was isolated using DNA-Extran 1 kit (ZAO Syntol, Russia). The test for genotyping was developed based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-PDRF) method. PCR amplification was carried out on a Bio-Rad T100 thermal cycler (Bio-Rad Laboratories Singapore) followed by RFLP analysis of amplification products to differentiate the SNP alleles. Specific endonucleases with restriction sites in mutant alleles were found using the NEBcutter v2.0 program (https://nc2.neb.com/NEBcutter2/). PCR-RFLP products were analyzed using 2 % agarose gel electrophoresis. The test system for the F94L MSTN polymorphism has been developed earlier (E.N. Konovalova et al., 2021). To verify the CAPN1 _316 amplification, Sanger sequencing was performed for three genotypes. In developing the diagnostic test for of Arg4Cys LEP и CAPN1 _316 polymorphisms, we used the sequences of NM_174259 and AJ512638.1 (NCBI, https://www.ncbi.nlm.nih.gov/). The polymorphism F94L MSTN CC genotype occurred in 98.77 % of investigated animals. For Arg4Cys LEP polymorphism, CT genotype prevailed with a frequency of 42.19 % for bulls and 45.68 % for heifers. For CAPN1 _316 polymorphism, the GG genotype predominated and accounted for 51.56 % of bulls and 69.14 % of heifers. The frequencies of the desirable productivity alleles among bulls and heifers were 0.00 and 0.01, respectively, for А F94L MSTN , 0.49 and 0.51 for С Arg4Cys LEP , and 0.20 and 0.28 for C CAPN1 _316. The bulls with Arg4Cys LEP TT genotype demonstrated the highest efficiency of live weight gain from birth to 8 months, the 778 g per day which is significantly higher ( t = 2.18) compared to CC (748 g). For CAPN1 _316 polymorphism, in heifers from birth to 8 months, there was a trend to higher daily weight gain in animals of CC genotype, 770 g vs. 720 g for GC genotype and 730 g for GG genotypes. However, in the post-weaning period, the observed trends changed. The 12-month-old bulls with the Arg4Cys LEP CC genotype had a significantly higher live weight compared to CT bulls, The CAPN1 _316 GG heifers of 8 to 15 months of aged showed a significantly higher live weight gain compared to CAPN1 _316 CC , 790 g vs. 740 g. The developed test systems ensures detection of the Arg4Cys LEP and CAPN1 _316 polymorphisms and can be used for genotyping and selecting beef cattle of desirable genotypes