| Pedigree records of livestock are an important basis for seed selection and population genetic parameter evaluation.At present,because genealogical records is not complete and even miss in the sheep breeding farms,and this have an important impact on the subsequent assessment of heritability,the prediction of breeding values and the formulation of seed selection and mating schemes,the application of paternity test technology to identify individual and modify the error pedigree information have an extremely important practical significance.Microsatellite molecular markers have been widely used in animal genetic map construction and family management due to their characteristics of codominance,stability and high polymorphism.At present,most sheep microsatellite repeat units are 2-base repeats,which makes it difficult to accurately read alleles due to the shadow bands in PCR amplification,which often leads to unstable experimental results.In this study,the Small-tail Han sheep and Dorper sheep were the main research objects,we screened for four-base repeats microsatellite sites with high polymorphism based on the sheep reference genome sequence,and explore the feasibility of the obtained microsatellite loci in sheep paternity test,this will lay the foundation for sheep genetic diversity analysis,paternity analysis and pedigree analysis.The main results of this papers as follows:(1)We obtained 138 microsatellite with four base repeats(ATAG or CTAT)_non 26 autosomals by BLAST retrieval in sheep reference genome.,and 53 sites were selected to design primers according to the characteristics of flanking sequence and the basic conditions for designing primers.By agarose gel electrophoresis and PCR product cloning and sequencing,the results showed that 30 of the sites had good PCR amplification specificity and amplification efficiency,the(ATAG or CTAT)repeat number range from 6 to 26,and the amplification length range from 127 to 468 bp.(2)The polymorphism of 30 microsatellite loci was detected with 31 small tail cold sheep.The genotyping results showed that 253 alleles were amplified from the 30 markers,the average number of alleles was 8.433,the number of alleles were all greater than 5,the polymorphic information content(PIC)vaired from 0.566 to 0.898,the calculated observed heterozygosity(Ho)ranged from 0.548 to 0.903,the expected heterozygosity(He)ranged from 0.631 to 0.921,with a mean of 0.7762.The Hardy-weinberg equilibrium analysis revealed that all 30 sites were in genetic equilibrium.(3)We randomly selected 11 microsatellite loci in 21 Dorper sheeps for polymorphism analysis,the results of allele typing showed that a total of 58 alleles were amplified,and the average number of alleles was 5.273.The PIC varied from 0.412 to 0.814,with a mean of 0.5688,the calculatedHovaried from 0.368 to 0.789,theHevaried from 0.477 to 0.855,with a mean of 0.6363.(4)In the paternity test site screening,22 microsatellite sites were first selected from the 30 polymorphic sites for the calculation of paternity exclusion probability,according to the size of the PIC from high to low in order to increase the number of locus.The results showed that when the genotypes of the two parents were unknown,the cumulative probability of exclusion(CPE)can reach more than 99.99% when the number of marker sites was 15,and theNe-1Pvaried from 0.321 to 0.663.(5)According to the size of the PIC from high to low to selected five combinations of 9,12,15,18 and 21 sites from 22 microsatellite markers for parental analysis in 4 sheep families.The results showed that when the number of marker sites was 9 and 12,some of the candidate fathers could only achieve low confidence or could not determine the parent-child relationship.And when the number of marker sites was 15,18 and 21,the candidate parents reached high confidence,and the results were completely consistent with the genealogy records,which laid a foundation for the sheep paternity test and the construction of molecular genealogy. |
PDF Full Text Request