Research And Application Of Potato Variety Authenticity Molecular Identification Technique

In order to more efficiently and accurately carry out authenticity identification of potato varieties,in this study,twenty pairs of SSR primers with high polymorphism and stable amplification were screened by non-denaturing polyacrylamide gel electrophoresis and fluorescence capillary electrophoresis,while it could determine the characteristic peak identification and reference varieties for each primer;The genetic diversity and population structure of the 231 samples that were collected and preserved were studied systematically,the while unique fingerprints were constructed for the samples tested;In addition,the authenticity of twenty resource materials was identified by using the selected primer combination,and the detection results of non-denaturing polyacrylamide gel electrophoresis and fluorescent capillary electrophoresis were compared and analyzed.The main research results were as follows:1.Thirty-four backbone samples were analyzed by using the non-denaturing polyacrylamide gel electrophoresis,while twenty-eight pairs of SSR markers that with clear amplification bands and good repeatability were preliminarily selected from eighty-one SSR markers.The selected primers were labeled with fluorescent dyes,while the ABI 3730 electrophoresis instrument was used to analyze the 231 tested materials by four-weight fluorescence capillary electrophoresis,and twenty pairs of primers with strong polymorphism and easy-to-read peak type of capillary electrophoresis were selected.By using the correlation analysis of different primer number combinations and the comparative analysis of the correlation coefficient between the genetic similarity coefficient matrix with different primer number and marker number,which could be seen that the twenty pairs of primer combinations screened were the minimum primer combinations for the genetic diversity analysis of231 samples.The peak type characteristics of each primer in capillary electrophoresis were analyzed,and the characteristic peaks of each primer were identified,so that it could ensure the consistency and accuracy of the read data.Forty-eight samples were selected as reference materials to correct the systematic errors.The materials with the same name as ‘Zhong Shu 3’ and ‘Zhong Shu 5’ were selected for authenticity identification with the standard samples stored in the resource library.The results showed that the fingerprint data of the sample to be tested and the standard sample were consistent,and it was determined that it was the same material as the standard sample.2.Twenty SSR markers were used to analyze the genetic diversity of the tested materials,while the 211 allelic mutations were amplified in 231 materials,and the logarithm of material that with genetic similarity coefficient in the range of 0 to 0.5000 were accounted for 1.4% of the total,also the genetic diversity was not abundant.Using the adjacency clustering analysis that based on the genetic diversity coefficient could divide the test materials into three large groups,and the algorithm that based on the Structure software model also could divide all materials into three subgroups,in addition,the results of clustering analysis and group structure analysis showed that most of the materials with consanguinity were clustered together.The selected 20 mark combinations were used to construct 231 pieces of material-specific fingerprint data,and all the materials could be distinguished.3.Using the electrophoresis capillary electrophoresis and non-denaturing polyacrylamide gel electrophoresis,while twenty labeling combinations were used to analyze the electrophoresis results of the twenty materials respectively.Compared with the results of fluorescence capillary electrophoresis,which was found that 70 more allelic variants were detected by non-denatureing polyacrylamide gel electrophoresis than fluorescent capillary electrophoresis,while the primers PIC detected were also higher,but the clustering results of the two had a certain similarity.The fluorescence capillary electrophoresis could be used to read out the allele variation size by software,while the detection results were more rapid,accurate and easy to be analyzed statistically.The twenty pairs of primer combinations selected were used to construct fingerprints of twenty materials for authenticity identification,while the results showed that the number of difference sites between the two materials ‘C58’ and ‘C60’ was equal to 0,therefore,it could be a suspected breed,the field identification results also confirmed the molecular identification results and the DNA fingerprint information of the varieties could be used as the molecular basis for the inter-species identification.This study based on fluorescent capillary electrophoresis and non-denaturing polyacrylamide gel electrophoresis for core primer screening,peak identification,and statistical analysis of fingerprint data provides important evidence for improving the SSR marker method in potato authenticity identification,and is conducive to further regulating the seed industry market.To protect the legitimate rights and interests of enterprises,breeders and farmers;analyze genetic diversity and population structure of the test materials,reveal the genetic background of the test materials,and provide theoretical basis for the collection of potato germplasm resources and the selection and breeding of new varieties.