| Fish is an important source of high-quality protein,rich in vitamins and minerals,good taste,easy to digest and absorb the advantages.Yellow River carp is famous at home and abroad for its golden scales,red tail and delicious meat,and it is a precious fish resource.The establishment of traceability system of Yellow River carp can realize traceability management of Yellow River carp products,so as to provide authenticity guarantee for consumers and strengthen the government and enterprises'supervision on meat product quality and safety.Due to the influence of artificial breeding and environmental changes,it is very difficult to use traditional morphological methods to identify individual varieties,which often cannot be fast and accurate.Moreover,it is impossible to identify individual varieties once the products are made and no longer have appearance characteristics.DNA markers are characterized by simple operation,mature typing methods,fast identification speed and high accuracy.They can be applied to trace the source of meat products,and can be used in all links of production,processing,storage and sales,and are not affected by biological morphology and status.However,at present,there are many difficulties in the application of DNA markers in traceability,including the selection of markers,mature and simple typing techniques,and data analysis in the actual application process.Therefore,in view of the above problems,this study carried out the research and application of the traceability of Yellow River carp based on SNP markers,laying a foundation for the application of DNA markers in carp species traceability.Specific content and research results are as follows:1.A total of 6 major carp species in China were collected,including 70 samples of Huanghe carp,50 samples of Hebao red carp,Furui carp,Songpu jing carp,Jian carp and Feng carp,and all samples were identified according to the corresponding standards.Two methods of database screening and trait gene search were used to screen SNP markers for Yellow River carp traceability.First,54 candidate marker sites were selected according to the Carpbase database search(Biotechnology Research Center,Chinese Academy of Fisheries Sciences),and multiple sequencing primers were designed for the candidate sites.The primers were used to amplify 170 samples,and the second generation of products was sequenced for population verification,and 42 SNP sites were detected.According to allele frequency and genotype frequency of the above loci in each variety,19 specific loci were finally obtained.Secondly,site search was conducted by trait genes.Through NCBI and Carpbase database retrieval and literature report,53 genes related to carp body color,growth traits and meat quality traits were selected,and 430 pairs of multiple PCR primers were designed for the detected fragments.Finally,111 pairs of primers were optimized to perform second-generation sequencing on the DNA target regions of 130 samples.The obtained SNP sites were screened according to the database method,and 19specific markers were obtained.Twelve SNP markers with the best specificity were selected from 38SNP candidate loci and divided into five groups for the identification of Huanghe carp and other five varieties respectively.The identification effect of Huanghe carp was the best with Songpujing carp and Fengquan carp,with MP value close to 0.Yellow carp was better than Hebao red carp,Furui carp and Jian carp,with MP value ranging from 1×10-5 to 6×10-4.The combination specificity of the 12 SNP loci was high.The MP values of Huanghe carp and other varieties were all less than 6×10-4,indicating that the identification effect of Huanghe carp was good.2.Establishment of SNP typing detection method.Using allele specificity,universal primers,mutated sequence primers and wild sequence primers were designed.The products were detected by capillary electrophoresis,and the peak location was determined quickly.The results were compared with the second generation sequencing.The results show that the method is consistent with the second-generation sequencing results of the corresponding region.The sensitivity of the method was tested by 10-fold dilution method.The sensitivity of the method was about 1.39ng/?L,and the results showed that the method was effective.The relative standard deviation(RSD)of the concentration of the five products was 22.88%,which indicated that the method had good reproducibility.A SNP typing method based on AS-PCR-CE was established,which combined the advantages of simple AS-PCR and specific amplification with the advantages of high efficiency,high sensitivity,high throughput and simple data analysis of capillary electrophoresis.3.The obtained 11 SNP markers were divided into five groups for the traceability identification of Yellow River carp and common carp.The AS-PCR-CE method was used for the determination of 300common carp samples.Among the 70 Yellow River carp samples,69 of the 70 Yellow River carp samples had peak in all 5 groups,and the number of groups with one peak was 4.The misjudgment rate of Yellow River carp was 1.43%.The number of groups of 230 common carp out of peak was less than or equal to 4,and no one was wrongly judged as Yellow River carp,and the accuracy was 100%.In addition,we also collected 5 samples from the Yellow River carp in Zhengzhou section,Tai'an section,Beijing Sidaokou Yellow River carp and Tangshan,Hebei,respectively,for authenticity identification.The results showed that there were peaks in 5 groups of carp samples from Zhengzhou section of the Yellow River,and all of them were genuine carp.There were 4 samples and 5 groups of Tai'an Yellow River Cyprinus carpio out peak,which were the genuine Yellow River Cyprinus carpio.Beijing Sidaokou Yellow River Cyprinus carpio has 2 samples and 5 groups all out peak,is the Yellow River Cyprinus carpio genuine product;There were 3 samples and 5 groups of Huanghe carp in Tangshan,Hebei,which all showed peaks,and they were genuine Huanghe carp.6 named the Yellow River carp for other carp counterfeit,counterfeit rate of 30%. |
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