Study On DNA Fingerprint Of Vitis Vinifera Resources Based On SSR Molecular Markers

The theme of this topic is Vitis vinifera resources protection and wine quality control,collecting 10 Vitis vinifera varieties in wine producing area on the north slope of Tianshan Mountain,Xinjiang.First,the genome DNA of Vitis vinifera are extracted by improved CTAB method,then the better polymorphic SSR primers are selected,and the SSR-PCR system is optimized and the electrophoresis spectrum is established.Finally,the genetic diversity of Vitis vinifera and its molecular identity code were established,which provided scientific reference for protection and identification of Vitis vinifera resources.Meanwhile,10 Vitis vinifera varieties were brewed,and then the flavor compounds were detected.The aroma characteristics and differences of wine were analyzed.The main results are as follows:(1)An improved CTAB method based on Vitis vinifera characteristics and two commercial kits were used to extract the genomic DNA of sample.The qualities of DNA was detected by agarose gel electrophoresis and nucleic acid protein analyzer.The results showed that the OD₂₆₀/OD₂₈₀ of DNA extracted by the improved CTAB method could reach 1.8,and the agarose electrophoresis strip was clear;The OD₂₆₀/OD₂₈₀ of DNA extracted by A kit was less than 1.8,the strip was clearer,but there is a bright spot at the entrance of the sample.It indicates there be exist other large molecular impurity contamination.The OD₂₆₀/OD₂₃₀ of DNA extracted from B kit was less than 2,the strip is blurred and the trailing phenomenon appears,which indicates the quality of DNA is not high and contains impurities.Compared with the above extraction effect,the best improved CTAB method was selected to extract DNA,and then the DNA of the sample was used as the template to the PCR amplify of the plant endogenous gene 18S r DNA.Electrophoretic detection showed that all samples could amplify PCR bands,which were clear and bright.It shows that the quality of DNA extracted from the optimized CTAB method has reached the requirements of subsequent molecular biology research.(2)The total DNA of Vitis vinifera was taken as a template,and the SSR-PCR amplification system was optimized.By combining single factor and orthogonal experiment,5 main components of the amplification system were optimized,and the optimal PCR system of 25?L was finally obtained.The results showed that the SSR-PCR optimal 25?L reaction system optimization for establishment of samples orthogonal test for Mg²⁺concentration of 2.5 mmol/L,d NTPs concentration of 0.15 mmol/L,Taq DNA polymerase concentration of 1.5 U,primers concentration of 0.5?mol/L,DNA concentration of 40 ng/25?L.The main components of PCR system were obtained by the visual analysis and variance analysis of optimization experiments.The influence degree of amplification was Mg²⁺>Taq DNA polymerase>d NTPs>primers>template DNA.The system was used to amplify the SSR-PCR system and then be detected by the non denatured polyacrylamide gel electrophoresis.The results showed that the band was clear and stable,which indicated that the system could be used for the study of SSR markers in Vitis vinifera.(3)SSR marker analysis technique was used to analyze the relationship between red Vitis vinifera and white Vitis vinifera and establish the molecular identification of the samples by the non denatured polyacrylamide gel electrophoresis atlas.The SSR primers with better polymorphism and reproducibility were selected for capillary electrophoresis detection.The results showed that twelve pairs of polymorphic primers were used for SSR amplification.213DNA fragments were generated and all of them were polymorphic bands.The genetic diversity analysis of SSR amplification results were analyzed by NTSYSpc2.10e software and the genetic similarity coefficient was 0.71 to 0.85 with the average genetic similarity coefficient of 0.76.The clustering dendrogram constructed by unweighted pair-group method with arithmetic mean(UPGMA)method indicated that 10 samples could be disaggregated two groups in the similarity coefficient of 0.73.This study showed that SSR molecular marker analyzed the genetic diversity of different kinds of Vitis vinifera from the molecular level and revealed the relationship between different materials.After alleles assignment,there were only 6 pairs of primers selected out for the construction of germplasms molecular ID.Meanwhile,5 pairs of primers with good polymorphism and reproducibility were selected for capillary electrophoresis,and the SSR locus fluorescence map of the samples was established.This provided a reference for identifying grape varieties and detecting the authenticity of wine.(4)SPME technology was used to extract volatile aroma components of wine.The aroma components of wine were detected by GC-MS and GC-O-MS,and the aroma characteristics and components of the wine were analyzed.The results showed that 10 varieties of wine were analyzed by GC-MS,and the volatile aroma components were mainly composed of 15 esters,9alcohols,2 acids,1 ketones,2 aldehydes and 2 phenols.GC-O-MS analysis showed that the 29aroma compounds were divided into 5 main aroma types:fruit aroma,flower fragrance,cheese flavor,grass flavor and almond flavor.The main aroma,flower and apricot flavor of variety wine have overlapping aroma volatile substances,of which all the samples have the isoamyl acetate,ethyl octane,benzyl alcohol and isoamyl alcohol,and the olfactory intensity is basically the same.Among them,Cabernet Sauvignon,Cabernet Franc,Merlot,Midknight Beauty and Petit Manseng smelled unique odor substance,but Cabernet Gernischet,Shiraz,Marselan,Chardonnay and Italian Riesling did not smelled unique odor substance.