International Journal of Scientific & Engineering Research Volume 9, Issue 10, October-2018 1907 ISSN 2229-5518 Variants mining of Kappa casein (K-CN) and Prolactin (PRL) genes among four indigenous cattle breeds in Nigeria Olanrewaju B. Morenikeji, Olawale J. Ogunshola, Mathew Wheto, Isaac A. Adebayo, Clifford A. Chineke Abstract The study determined the allele and genotype frequencies of genetic variants in two genes associated with milk production traits in four indigenous cattle breeds (N’dama, White Fulani, Muturu and Keteku) in Nigeria. Polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP) technique was used to detect genetic polymorphisms in two milk genes (kappa casein (K-CN) and Prolactin (PRL)). Three genetic variants (AA, AB and BB) of K-CN gene were detected in White Fulani and Muturu cattle breeds among the four breeds considered in this study. While the PRL gene was found monomorphic in all the sampled animals as they were all genotyped as AA homozygous PRL genotype. The detection of genetic polymorphism in White Fulani and Muturu cattle breeds revealed the following allele frequencies of kappa-casein gene A and B estimated as 0.69, 0.31 for White Fulani; 0.56 and 0.44 for Muturu respectively. The detection of both alleles A and B as observed in these animals established a high degree of genetic variability among the four cattle breeds for K-CN locus. The milk genes are polymorphic in their expression and there were genetic diversities in the cattle population understudy. Also, a higher frequency of A allele in the animal sample population which has been associated with unfavorable milk production and protein yield revealed the need to breed for increased B allele among the Nigerian indigenous cattle through genomic selection. Overall, the results of this study showed that breeding to improve milk production among indigenous cattle breeds are feasible through genomic selection. Keywords: Genes, Cattle, PCR-RFLP, frequencies, polymorphism 1 INTRODUCTION Selection process can take in to account the variations in Indigenous cattle in Nigeria serve as the backbone of animal’s performance that is related to the genetic relevant and sustainable cattle production because of better polymorphism. adaptation to survive and reproduce under harsh Reinhardt et al. (2012) stated that milk is an important environments though with low performance as compared source of essential nutrients for both lactating calves and to high performing exotic counterpart breeds. However, human food. Studies have shown that ruminant’s milk most Nigerian indigenous cattle are better meat producer protein genes are highly polymorphic with a large number but poor or low milk producer (Ezekwe, 2001). of unusual polymorphism (Nilsen et al., 2009). As far back In the past, animal breeders have made effective efforts to as 1955, Aschaffenburg and Drewry have discovered improve the production performance of livestock species variants A and B for β lactoglobulin in cattle which has by altering their genome through the selection of superior become a worldwide interest in genetic polymorphisms in parents for the next generation.IJSER This involved the direct use milk proteins. These variants are believed to be controlled of phenotypic records without the knowledge of the by the codominant autosomal genes which are according to molecular information. The outcome may improve the Mendelian inheritance. This study was corroborated with a production while ignoring some reproductive performance review by Caroli et al. (2009) who also identified variants in of the animal. Sometimes, attempts were made through milk protein. cross breeding of the indigenous animals with the exotic Milk genetic variants are different by few amino acid ones which has not been without difficulties like loss of substitutions or deletions within the polypeptide chain distinctive qualities such as disease resistance, heat (Eigel et al. 1984, Caroli et al., 2004 and D’Alessandro et al. tolerance, ability to survive and reproduce under stress and 2011). Milk protein genetic variability in cattle is necessary low feed input thus posing threat to the indigenous breed. at both the DNA and protein levels for evolutionary and Whereas, the advent of molecular techniques has made biodiversity analyses (Caroli et al., 2004). breed characterization a possibility by determining genetic relationships among animals based on differences in their The frequencies of genetic variants of milk proteins in DNA. Therefore, there is need to adopt methods of different cattle breeds and the possible relationships with selection that are based on genomic studies. (Karp et al., the milk production traits such as milk composition, milk 1996, André, 2012). quality and quantity have been widely studied due to their The molecular DNA markers have been deployed for breed possible use for milk protein characterization as a mean to characterization in both plants and animals. For example, genomic selection (Jeichitra et al., 2003; Caroli et al., 2004; marker-assisted selection have been practiced in dairy and Yasemin and Cengiz, 2006). cattle whereby some genes were identified as potential On the other hand, there is no available comprehensive candidates which are associated with performance traits in study of allelic variants in Nigerian indigenous cattle breed dairy cattle (Marson et al., 2005, Hassen et al., 2007). up till date. A study to identify different allelic variants in IJSER © 2018 http://www.ijser.org International Journal of Scientific & Engineering Research Volume 9, Issue 10, October-2018 1908 ISSN 2229-5518 milk genes, allele frequencies and their effects in 2.2 Genomic DNA extraction indigenous breed performance is important for Blood samples were collected from 300 animals for genomic improvement in Nigerian cattle breeds. Genotyping dairy DNA isolation through the jugular vein in vacutainers animal at the molecular level is not limited to cows alone. containing ethylene diamine tetra acetic acid (EDTA) and There is possibility to genotype males, non-lactating kept on ice to maintain low temperature in order to prevent females and embryos at the DNA level because milk cell lysis. Subsequently the blood samples were transported protein typing does not require the gene products (Caroli et to the laboratory and stored at 4oC prior to genomic DNA al., 2004). isolation. Genomic DNA was extracted from whole blood Polymerase chain reaction-restriction fragment length using standard procedures as recommended by the polymorphism (PCR-RFLP) based analysis is a popular manufacturer (Qiagen Inc., Valencia, CA) and the DNA technique for genotyping. Besides being valuable for the was stored at 4 oC until ready for use. determination of intraspecies variation, the PCR-RFLP technique is very popular for species identification and 2.3 Amplification of genomic DNA by Polymerase differentiation(Rojas et al., 2009). For example, Hassen et al chain reaction (PCR) (2007) used PCR-RFLP to assess genetic variability within Specific primer pairs (from Table 1) were used for each and among five indigenous Ethiopian cattle breeds. The reaction. A final volume of 25 µl reaction mixture was analysis revealed that within breed genetic variation was prepared with 2µl of genomic DNA (50–80 ng), 12.0 µl Taq much higher than that between breeds. The PCR-RFLP DNA polymerase master mix, 60ng of each primer, and 10 technique is quick, less expensive and requires less µl of nuclease free water. PCR-reactions were carried out in expertise which makes it to be easily adopted for molecular 0.2 ml of PCR reaction tubes using a programmable thermal characterization. cycler (BioRad, C1000 TouchTM) with cycling conditions as, The current study was designed to utilize PCR-RFLP initial denaturation at 94◦C for 3min, denaturation at 94◦C technique to identify genetic variants in two milk genes for 30 sec, annealing at 60◦C for 35sec, and extension at (kappa casein (K-CN) and Prolactin (PRL) with a view to 72◦C for 3min were carried out for 40 cycles followed by determining the allele and genotype frequencies of milk final extension at 72◦C for 10 min. After 40 amplification gene variants among four indigenous cattle breeds in cycles, the amplified products were verified by Nigeria and to make possible recommendation for genomic electrophoresis on 1.5% w/v agarose gel in 1×TAE buffer selection in place of the current tradition selection methods with 100bp ladder for 50mins. The gels were stained with for dairy cattle improvement in Nigeria. Ethidium Bromide and visualized under UV light by the gel documentation system (Enduro, Inc). 2 MATERIALS AND METHODS 2.1 Animal Sampling IJSER2.4 Variants identification through RFLP and agarose gel electrophoresis A total of 300 animals comprising 84 White Fulani, 78 In order to identify genetic variants among the sampled Keteku, 62 Muturu and 76 N’dama indigenous cattle were animals, 20 µl of PCR products were digested with 10 units randomly selected and genotyped. The animals were of the restriction enzyme (Invitrogen, USA) which is sampled from the Teaching and Research Farm, Federal specific for each gene (Table 1) in a final reaction volume 25 University of Technology Akure (FUTA), Federal College of µl. The reaction mixture was incubated at 37oC in heating Agriculture, Akure (FECA) and Institute of Agricultural blocks for 5hours and fragments were separated in agarose Research and Training (IAR&T), Ibadan. gel. Briefly, after restriction digestion the fragments were analyzed and separated through electrophoresis in 3% • Dr. Olanrewaju B. Morenikeji, Federal University of agarose gel which was stained with ethidium bromide and Technology, Akure, Nigeria ([email protected]) a 100-bp ladder was used as the molecular marker. • Msc. Olawale J. Ogunshola, Federal University of Technology, Akure, Nigeria ([email protected]) Thereafter, the gel was visualized by the UV gel • Dr. Mathew Wheto, Federal University of Agriculture, documentation system (Enduro, Inc). Abeokuta, Nigeria ([email protected]) • Professor Isaac A. Adebayo, Federal University of Technology, Akure, Nigeria ([email protected]) • Professor Clifford A.