Study On Population Genetic Diversity And Establishment Of Agrobactrium-mediated Transformation In Malus Sieversii

Malus sieversii is mainly distributed in Yili valley of Xinjiang,eastern of Kazakhstan and Kyrgyzstan,Tajikistan and other places of Central Asia,which is along with the Tianshan mountains.The merit properties of Malus sieversii is higher than domestic apple,such as insect resistance,disease resistance.It is the important apple stock resource of China’s western region,it is also a rare form of the tertiary relic species.However,the distribution area of Malus sieversii is decreasing with people’s activity,with the dangers of Agrilus mali Matsumura in recent years.So the research of population genetic structure and merit resistance gene will lay theory foundation for apple cultivated breeding.Our research group went Yili valley of Xinjiang including Xinyuan,Gongliu,Huocheng and Nalati,for investigation of growth condition,distribution area,agronomic characters of Malus sieversii,also,the related plant materials are collected to do experiment in April of2015.Simple Sequence Repeat Polymerase Chain Reaction was amplified and electrophoresis detection of Malus sieversii by setting different concentration gradient of all ingredient in the methods of control variable to chose the optimal reaction system of Malus sieversii SSR amplification.Thereby,we did a research on the genetic structure of Malus sieversii by SSR.Callus induction system was established using tissue culture seeding of Malus sieversii as material by set different hormone gradient.Cloning the merit resistant gene Ms USP of Malus sieversii from transcriptome data analysis through the homologous cloning,building the expression vector,using the method of Agrobacterium-mediated transformation to transfer the Malus sieversii and Arabidopsis thaliana,the main results are as follows:1)Phenological period of Malus sieversii in different areas is different,the population of Daxigou and Xinyuan are in full bloom in the same altitude,however,the population of Nalati represents in bud period stage.In the same area,the population in high altitude are late for flowering,with earlier flowering of low altitude.Filaments and ovary length of Malus sieversii is Gongliu >Xinyuan> Daxigou> Nalati in turn.Filaments and ovary length of M.neidzwetzkyanais is 8.69 mm and 5.617 mm,respectively,all of which is higher than Malus sieversii in length.2)According to the definition and polymorphism of amplified bands,the optimal reaction system of Malus sieversii SSR amplification was determined,the gradient of 10×PCR buffer,d NTP,Primer-F,Primer-R,Taq DNA polymerase,DNA templates is 2.5 u L,0.2m M,0.5 m M,0.5 m M,0.5 U,100 ng in turn.3)Take software POPGENE version 1.32 and NTSYSpc-2.10 e on the SSR for data analysis,it is concluded that the four group genetic consistency of Yili,Tacheng,Kazakhstan,Kyrgyzstan is between 0.8346 to 0.9969,Yili and Tacheng population is the most close in genetic relationship.The coefficient of genetic differentiation between the four groups is an average of 0.1351,the gene flow is 1.6006,namely,variation of Malus sieversii mainly comes from inside.4)Nei genetic diversity index,Shannon’s information index of Yili in Xinjiang province is from high to low in order for Xinyuan population> Gongliu population> Daxigou population> Nalati population> ancient area of Malus sieversii,genetic consistency of six groups is between 0.88 to 0.98,Gongliu and Daxigou is closest relative,genetic consistency between Xinyuan and ancient area of Malus sieversii is the lowest,0.8851,indicate that the differentiation between population were small,variation in the population is the main origin of differentiation.5)Callus induction system of Malus sieversii was established,which is MS + 2.0 mg/L TDZ + 0.5 mg/L NAA;we selected the Kan optimal concentration of 50 mg/L by experiment on Kan sensitivity.6)The resistance gene Ms USP origin from Malus sieversii was successfully cloned,and build genetic expression vector of Malus sieversii successfully.Transfer plant of Malus sieversii and transformed Arabidopsis thaliana was successfully obtained by using the method of Agrobacteriuma-mediated.