Establishment Of DNA Fingerprintmap And Analysis Of Genetic Diversity Among Beet Cultivar

One of the most major targets of seed quality testing is the authenticity and Purity of cultivar.Theimportant significance to research rapidly,simple and accurate method of molecular maker with beetvarieties identified during seed quality testing and granting the new variety authorigation.Thefingerprint was built for the sugarbeet varieties applied in the sugar beet production in our country.SSRmolecular marker used in this research first time,and various factors affecting the variety purity andauthenticity identification were analyzed to establish a set of quick and efficient method for the varietyidentifying, which would make it possible to interchange between different laboratories.The majorresults obtained as follows:1. An improved method of rapid,high effective DNA extraction was set up.In this study, Used dry beet seeds or leaves which treated1～2days with frozen and dried in thevacuum freezing machine and extract DNA,the PCR plates of96were used to instead of a singlecentrifugal tube,Alkali decomposition method was used for DNA extraction,and in stead of liquidnitrogen.one person can extract about100DNA samples in20minutes,DNA extracted to be used astemplate of SSR-PCR.2.29primers were screened as the core primer of SSR to apply in fingerprint constructionUsing8%non denaturing polyacrylamide gel to test101pairs of sugar beet SSR amplificationproduct, each experiment repeated once at least,29pairs of primers were found which with clear bands,good repeatability, high polymorphism and easy to identify, each pair of primers amplified out2-11alleles average, there were159alleles all together，including116polymorphic alleles,and the ratio ofpolymorphism was72.95%.The fragment sizes vaired from90-500bp.The29primers can be used ascore primers for sugar beet varieties purity and authenticity identification.3.Multiplex SSR-PCR amplification system of beet was establishedBased on the single-pair of primer SSR-PCR, Duplex or triplex SSR-PCR system for Beet is thatevery increase one plex of SSR, and the amount of the appropriate primers increases only and thecorresponding amount of deionized water reduced, and the remaining ingredients unchanged. Four orfive plex SSR-PCR of beet is on the basis of the single PCR to increase the amount of0.5times DNTPsand the corresponding primer, at the same time which is according to the different individual primers inamplification efficiency, a corresponding reduction of the individual primer in mutiplex PCRamplification system, In this system,most four or five plex PCR is effective. the detection efficiency is2～5times than the single-primer PCR.4. Seven pairs of primer can be used to identify different varieties independently, The DNA fingerprintsof46beet varieties were constructed by three primers。UPGMA cluster analysis of genetic distance showed that all of the varieties were clustered in onegroup at the genetic distance of0.20, In genetic distance0.16,46varieties were divided into four groups,Cluster analysis and the source of the material has fine consistency, varieties come from the samecompany or the same country basically to join together,it is indicated that the genetic basis of beetvarieties was narrow. In addition, there are seven pairs of primers SB04, S7, S6, BVV45、SB13、S13、 S8, which can be able to identify the different varieties independently, the primer of SB04, S6and S7will made distinguishi for all varieties.5. A rapid method to identify beet varieties purity and authenticity was establishedA96-well PCR plate was used which instead of a single PCR tube, alkaline lysis method was usedto extract form dry seeds (or dry powder) DNA; the GV (Goldview) and λ DNA were used to test DNAconcentration and quality; PCR reaction system is10μL; PCR reaction program cycle denaturation andannealing temperature were converted to the15s, and extends for30s,35cycles; the high resolution of8%non-denaturing polyacrylamide gel was used to separate the PCR product; finally, A rapid silverstaining was used to detect polyacrylamide gel. At the same time，we put DNA templates, primers,DNTPs and PCR Buffer into the refrigerator-fresh-layer, Don’t need to melt when using, all of themcan normally use within3months.In this way,we established a set of simple, fast, low costing and littlepollution technical system of the beet varieties authenticity and purity identification system, which is topromote the application of the technology in large-scale,A mature operator of laboratory can detect192samples by one day.