Applying Microsatellites DNA To Identify The Paternity Of Holstein

The objectives of this study were to test the genetic polymorphism of Holstein by Microsatellites DNA, and further to set a system of identifying the parentage for cattle.Microsatellites DNA (short tandem repeats, STR; simple repeat sequences, SRS; Simple Sequence of Repeats, SSR) is a special class of tandemly repeated DNA in which a specific motif of 1-6 bp is repeated up to 100 times. Most of the repeat sequences are two-nucleotide repeats, but there is also one-, three- or six-nucleotide repeats in the repeat sequences. The length of the microsatellites DNA are commonly <400 bp. Microsatellites loci with >10 repetitions of the basic motif are highly variable in taxa ranging from plants to vertebrates. Microsatellites DNA sequences have been shown to exhibit length polymorphisms. Based on the trait of length polymorphisms, it can be used to identify the parentage for animals. In this experiment, 12 microsatellites DNA loci recommended by FAO and ISAG were chosen to identify the paternity of Holstein:ETH10, ETH225, INRA023, INRA063, BM1818, BM1824, ILSTS006, CSSM66, HEL9, TGLA53, INRA037, TGLA126.Firstly the genetic polymorphism of Holstein with a sample of more than 100 cattle from different farms was examined using these microsatellites DNA markers by PCR and Electrophoresis-Argentation. The genetic heterozygosity, polymorphic information content and the effective number of alleles were all calculated. The result showed that the mean of the genetic heterozygosity, the polymorphic information content and the effective number of alleles of the 12 microsatellites DNA loci were respectively 0.8711, 0.8574 and 8.1666, the Cumulate Probability of Exclusion is 99.999999%, both the value of the heterozygosity and the polymorphic information content were higher so all of the 12 STR loci were high polymorphism and they could be used to the genetic analysis and individual identification in animal.36 Holsteins were tested and their biological father was identified using these 12 polymorphic microsatellites loci in different three conditions: 1. Given the genotypes of both parents and child are known, to exclude one of the parents. 2. Given the genotypes of both parents and child are known, to exclude both the parents. 3. Given the genotypes of one parent and child are known, to exclude their paternity. The results showed the 12 STR could identify the biological paternity of the cattle in three different situations, so they can be used in parentage test.Secondly four sets of multi-PCR amplification systems were optimized. The sets were as follows: Set1: ETH10-BM1824-ETH225 Set2:INRA063-TGLA126-ILSTS006 Set3: ILSTS006-THLA53-INRA037 Set4: INRA063-HEL9-TGLA126The genetic polymorphism can be analyzed by multi-PCR, but because most of the length of microsatellites DNA is between 100 and 300 bp, and with its high polymorphism, quite a few of non-specific bands appeared or one or two specific bands were diminished when the multi-PCR results were tested by PAGE compared with single PCR result. So the result was that multi-PCR amplification systems would be more suitable for fluorescence-Mutiplex PCR in the identification of individuals. If fluorescence-primers were not available in the experiment, single PCR amplification system is more suitable for the parentage identification.It is generally considered the causes of the polymorphism in microsatellites DNA are the sliding, mismatching of the bases when DNA is copying and repairing, or the asymmetrical division between the sister chromatids during the period of meiosis. The short tandem repeats of the locus, ILSTS006 were also tested by DNA sequence analysis. Different genotypes of the locus were sequenced and the results were consisted with the theorical values based on its repeat numbers.