Evaluation of microsatellite markers for paternity testing in cattle

Abstract

A study was undertaken to evaluate the efficiency of microsatellite markers for paternity testing in cattle of Kerala. Genomic DNA was isolated from whole blood, fresh and frozen semen samples using phenol: chloroform method. DNA samples from 100 genetically unrelated animals were used to determine the polymorphisity of the markers and samples of known pedigree was used to test the inheritance of markers. The mean yield of DNA obtained from 5 ml of whole blood was 388.2 ± 14.3 µg, from fresh semen was 181.15 ± 6.2 µg/400 million sperms and from frozen semen was 116.95 ± 25.2 µg/150 million sperm cells. The optical density ratios (260/280) ranged from 1.64 to 1.81, 1.42 to 1.73, 1.54 to 1.76 and for DNA obtained from blood, fresh and frozen semen respectively. Three microsatellite markers viz., DRB3, ETH131 and FSH out of a panel of tested markers were chosen for the study based on their polymorphicity and ease of typing. The forward primer of each primer pair was end-labelled with  32P-ATP. PCR parameters varied between the primers with respect to annealing temperature (60°C for DRB3 and FSH; 55°C for ETH131) and MgCl2 concentration (1.25 mM for DRB3 and FSH; 1.5 mM for ETH131). The amplified products fractionated by denaturing polyacrylamide gel electropheresis were visualized by autoradiography. The number of alleles was counted and allele sizes assigned by comparison with sequences of M13 DNA run along with PCR products. The frequency of each allele was worked out. Seventeen alleles with sizes ranging from 138-192 bp were identified for DRB3, 11 alleles of size ranges 134-168 bp for ETH131 and nine alleles of size ranges of 184-214 bp were observed for FSH. The heterozygosity values obtained for each locus were 0.8938, 0.8385 and 0.8519 for DRB3, ETH131 and FSH respectively. DRB3 was highly informative with PIC value of 0.8864 followed by FSH (0.8392) and ETH131 (0.8151). The probability of exclusion of incorrect sire was calculated independently for the three markers and the values were 0.7913, 0.6787 and 0.7035 for DRB3, ETH131 and FSH respectively. The combined probability of exclusion obtained with DRB3 and ETH131 was 0.9329 and DRB3 and FSH was 0.9381 and that with ETH131 and FSH was 0.9047. The three markers together yielded a cumulative exclusion probability of 0.9801. Thus the exclusion probability was found to increase with the number of markers. The inheritance pattern of these markers was tested on known sire families. All the three markers agreed with each other in identifying the correct sire and excluding the incorrect one. Though the efficacy of the three markers for paternity testing was found satisfactory, it was concluded that one or two similarly polymorphic markers have to be used along with the markers studied to obtain maximum probability of exclusion of 0.99.