Study On The Applications Of SNP And SSR Markers In The Traceability And Identification Of Beef

Traceability is viewed as the one of the most reliable and trustworthy methods to protect food safety and consumers’rights.So far,animal ear tag and product label is the main method to realize the traceability of aninal and its produect,however the traditional ear tagging system and paper documents are easily lost and replaced,they can not ensure the truth of the product and can not well trace in the whole food supply chain from a farm to a retailer through different operations.DNA-based genetic traceability can effectively compensate for the traditional ear tagging system and paper documents.In either raw meat or related products,DNA is never lost even through multiple processing steps,and could facilitate seamless connectivity in the whole food supply chain.So this study had built a genetic tracing system based on individual identification to trace the meat with SNP and SSR,providing a basis for the practical application to some extent.The main contents and results of this work are as follows:1.A device for simultaneously collecting blood samples and recording information was developed.The Whatman 903#paper was the optimal carrier paper for blood collecting,which could well store blood samples for traceability use.The paper carrier was wrapt with 0.125mm hot framed membrane for protection and the whole thing was fastened to the pillar of animal ear tag.In order to improve the success rates of the blood samples collected concurrently with tagging and do not cause excess hurt to the wound,the nail of animal ear tag was properly modified,including adding screw thread around the nail of animal ear tag and wedge-shaped gaps on the protruding edge of cusp of the nail.2.SNP loci for cattle individual identification and meat traceability were selected.First,a total of59 SNP markers were selected from the NCBI dbSNP database,with the next-generation sequencing on Illumina X-10 genotyping platform within 176 individuals belonging to seven cattle breeds,36 highly polymorphic loci with the MAF greater than 30%was obtained.When the 12 most polymorphic markers were chosen,the matching probability value was 2.6 out of million.Second,a total of 61 SNP markers were discovered from the 30 genes relating to growth and meat quality trait by Sanger sequencing,and the polymorphism and individual identification capacity need further test and verify.3.Two kinds of SNP genotyping methods were developed.One of them was AS-PCR-CE method,the sample was amplified by AS-PCR and the amplicons were separated by capillary electrophoresis,through the corresponding product appearing or not to confirm the genotype of one sample.And the method could accurately genotype the concentration of DNA template was 0.4ng/μL.The other method was a fast and visual SNP typing method basing on collaurum and RPA.The method could realize the exponential amplification of the target product under the 37℃within 30 minutes,and the sensitivity of method was 14ng/μL.4.SNP markers were employed to individual identification and meat traceability in practice.With the 192 individuals belonging to six cattle breeds,the 12 most polymorphic markers from meat and growth related gene were chosen,and the matching probability value was 2.6 out of 10~5 according to the result.Through the macthing of genotype barcodes between the meat and the blood from the individual to verify the truth of the traceability system.5.SNP markers were used to distinguish the meat from yak or cattle.Ten SNP markers from meat related genes were chose according to the allelic frequencies,and the allelic frequencies of the ten SNP loci was 0.546-0.833 in cattle population,and the allelic frequencies was 0.009-0.075 in yak samples.And one positive control primer set was selected.The multiplex allele-specific polymerase chain reaction and capillary electrophoresis method was employed for SNP genotyping.According to the number of amplicons,the samples were identified as yak or cattle meat.Yak samples generally produced one to three amplicon peaks,and the cattle samples yielded five or more peaks in the electropherogram.This method could be useful to distinguish yak from cattle,and it was suitable for processed meat.6.SSR markers were employed to individual identification and meat traceability.Sixteen markers were selected from the recommendation of ISAG and FAO,the fluorescence-multiplex PCR combined with capillary electrophoresis was used to genotyping within 100 individuals belonging to six cattle breeds.There were 15 polymorphic loci in the total population.When the 6 most polymorphic markers were chosen,the matching probability value was two out of million,the distinguishing effect of which was comparable to the use of 12 SNP loci.The results of PLS-DA indicated that the six cattle breeds were relatively well distinguished by the sixteen markers.