Establishment And Application Of DNA Barcode Based Methods For Molecular Identification Of Berries And Their Products

With the improvement of people's living standards,berries are known as the third generation of golden which are popula.However,the price of berries is often high due to high demand,low yield,short picking period and difficulty in storage and transportation.As a result,the adulteration and falsification of such berries and their products have gradually increased,causing consumers to question and worry about the quality and safety of related foods.At present,there are many researches on the identification and detection methods of berries and their products,mainly based on spectral technology,chromatography,mass spectrometry,electronic tongue technology,ultrasonic technology,molecular biology technology and so on.In this study we established a DNA barcode technology which can be used to identify the authenticity of berries and their products by using modern molecular biology technology,and tried to detect the berry products on the market.The main result are as follows:A better extraction method and DNA barcode amplification system for genomic DNA of berries were established.the results of electrophoresis and nucleic acid concentration and purity analysis showed that the improved CTAB method was suitable for berry genomic DNA extraction.By comparing the DNA amplification efficiency of five PCR amplification kits for different berry species,it is proved that KAPA HiFi Hot Start Ready Mix and 2 × Ulta HiFidelity PCR Mix have better amplification effects.Based on comprehensive economic considerations,KAPA HiFi Hot Start Ready Mix is selected as an amplification kit.2.Established a berry DNA barcode identification system.With cherry,dragon fruit,red raspberry,grape,mulberry,Chinese cherry,apple,blueberry,peach,passion fruit,coriander,pear,kiwi,blackcurrant,red grapefruit,cranberry,apricot,red pomegranate Eighteen kinds of fruits were used as test materials.The genomic DNA was extracted and the plant-derived 18S rRNA gene real-time fluorescence PCR method was used to verify that the quality of berry genomic DNA met the template requirements.Thirteen pairs of universal primers were used for PCR amplification of 18 kinds of berry genomic DNA.The products were verified by agarose gel electrophoresis to be ITS2,rbcL-1,rbcL-2,rpoC-2,psbA-trnH,psbA-trnK?trnH-psbA-1 amplification effect is better.Through one generation sequencing and BLAST alignment,the results proved that rbcL-2,ITS2,and psbA-trnH are the most suitable DNA barcode sequences for identifying berry species.3.Established a berry adulteration model system and its identification method.Cherries,blueberries,kiwi,grapes,apples,and pears were selected as test materials to quantitatively extract genomic DNA.The experiment designed six adulteration models of cherry seed blended with grape,cherry seed blended with pear,blueberry blended with grape,blueberry blended with pear,kiwi fruit blended with pear,kiwi fruit blended with apple,and genomic DNA level adulteration was used to estimate raw material level adulteration.The universal primers with the lowest detection limit were screened out to establish a method for identifying the authenticity of berries.Through PCR reaction,product electrophoresis,sequencing and sequence alignment,the test results show that:the universal primer ITS2 and psbA-trnH can be used for adulteration of the cherry seed model;the universal primer ITS2 and rbcL-2 can be used for adulteration of kiwi model;Universal primers psbA-trnH and rbcL-2 can be used in blueberry adulteration models to establish three identification methods for authenticating berries.4.Application of DNA barcode identification method in the detection of commercially available berry products.Using the established DNA barcode identification method,a total of 50 samples of commercially available berry juice,pulp,dried fruit and jam were identified based on ITS2,psbA-trnH DNA barcode.The results showed that among the 50 samples,70.00%were found to meet the label value,20.00%were found not to meet the label value,and 10.00%were found to be invalid.The above results show that the DNA barcode technology is suitable for authenticity identification of commercially available berry products,and provides a new idea for the detection of berries and their products.