Study On Identification Of Hops Varieties And Purity Based On SNP-KASP Technology

Hop is one of the basic raw materials for beer brewing.It plays the role of foaming and antiseptic in beer.It is the main source of bitter substances in beer and be called "the soul of beer".At present,there are many varieties of hops available on the market,and there is a phenomenon of inferior packaging.This not only affects the healthy development of the hop industry,but also has a greater negative effect on beer brewing companies.More importantly,this behavior may affect the health of consumers and cause food safety problems.However,the current hop national standard testing only involves four physical and chemical indicators,does not contain hop purity and authenticity testing.Therefore,it is imperative to develop an accurate,rapid and low-cost method that can be used to test the purity and authenticity of commercially available hops.This study aims to develop a set of technical means that can be applied to the identification of hop varieties,so that beer companies can effectively control the quality of raw materials for brewing,provide certain supervision methods for hop raw material suppliers,and provide product quality for beer brewing companies.The stability and the healthy development of the hop industry are escorted.This article used 18 kinds of hops used by domestic beer brewing companies as raw materials,based on SNP(Single Nucleotide Polymorphisms)single nucleotide polymorphism,used KASP(Kompetitive Allele Specific PCR competitive allele-specific PCR)Genotyping detection technology for hop identification and purity testing.First,hops genomic DNA was extracted.Then,through selecting high-throughput sequencing and bioinformatics analysis of 8 kinds of hops commonly used at home and abroad,the SNP sites that can be used for identification of hops were screened.Then,using the DNA of 18 hop varieties as a template,the SNP loci were verified by PCR(Polymerase Chain Reaction)amplification and one-generation sequencing,and SNP loci that could distinguish 18 hop varieties were screened.Finally,Based on the screened SNP loci and KASP genotyping detection technology,qualitative and quantitative analysis of premixed samples and corporate samples were performed,and the detection of premixed samplesand the identification and purity testing of corporate samples were completed.The feasibility and accuracy of this technology.The results are as follows:(1)Extraction of hop genomic DNA used the QIGEN kit.The results showed that the extracted genomic DNA fragment was more than 15 KP and the genome was complete.Concentration above 215 ng/?L meets experimental requirements.After the high-throughput sequencing and bioinformatics analysis of eight varieties of hops genomes,a total of 46,003 more reliable SNP loci were obtained.Through further screening,2636 SNP sites that can be used for species identification analysis were identified.(2)After the verification of authenticity and further screening,it was determined that11 SNP loci can distinguish 18 hop varieties,18 SNP data tables of hop varieties were obtained,and a road map for differentiation was established.(3)The premixed samples were detected by using 11 SNP loci.The results showed that the genotypes of the 8 sites of H01,H02,H03,H04,H06,H09,H10 and H11 were completely in line with expectations.When the three loci of H05,H07,and H08 were detected with samples over 5%,the results were consistent with that of premixed genotypes,but there was a deviation in the results of genotype detection in the samples with the degree of confounding of 2%.The results showed that the method can accurately distinguish the samples with a mixture ratio of more than 5%.In addition,because there was a very significant linear correlation between the two fluorescence signal ratios of KASP and the proportion of hybrid samples,the mixed ratio of different hybrid samples can be used as a standard curve to detect the purity of hops.Finally,used this technology to test Qingdao Dahua sample and Sazz sample sent by companies,the purity of Qingdao Dahua sample was more than 95%,while Sazz hop was a mixed sample.The purity of the Sazz hop was determined to be only 65.75% by the standard curve method.Compared with corporate information(Qingdao Dahua purity was more than 98%,Sazz hop purity was70%),the detection error was less than 5%.The above results showed that the SNP-KASP technology can effectively identify the variety and purity of hop samples.