Analysis Of Molecular Genetic Structure Of Natural Population And Infective CDNA Clone Population For Alfalfa Mosaic Virus

Alfalfa has many advantages,such as high yield of grass,wide planting area and rich nutrition,but with the growing of planting area,the diseases and pests of Alfalfa become more and more serious,especially the disease of alfalfa virus,it has a great effect on the yield and quality of Alfalfa.The Alfalfa mosaic virus(AMV)is a very widespread virus that infects more than 430 plant species and has a severe impact on many cash crop and legume species.In this paper,the natural population and infectious clone population of AMV were studied,the study covered the molecular genetic structure of natural population of AMV and the distribution of Alfalfa mosaic virus disease in different provinces of China,as well as the pathogenicity and adaptability of AMV to different hosts.It provides theoretical and technical support for the early warning and control of Alfalfa Mosaic Virus Disease in China.In the aspect of natural population research of Alfalfa mosaic virus,111 suspected alfalfa virus disease samples were collected from different alfalfa ecological production areas in China,and nine viruse species infected alfalfa were identified,AMV,To MV,CPMV,TMV,BYMV and CMV were detected infecting alfalfa virus diseases.At present,except Ganzi,Sichuan,other alfalfa producing areas are infected by AMV.In order to clarify the occurrence of Alfalfa virus diseases in China,the annual positive rate of AMV was tracked and it was found that the incidence of Alfalfa virus diseases caused by AMV was increasing year by year.In addition to AMV infection,other viruses were found in alfalfa in the following areas: BYMV infection in Langfang,Hebei Province;CPMV infection in Ganzi,Sichuan Province;BYMV,TMV,CPMV,CMV and To MV infection in Lanzhou,Gansu;TMV infection in Minhe,Qinghai;BYMV,TMV and SMV infection in Qiqihar,Heilongjiang.The results also showed that coinfection of these viruses in alfalfa were happened in these areas.In addition,phytoplasma infection was found in Cangzhou,Hebei and Qiqihar,Heilongjiang.It is the first time for the investigation of the distribution of alfalfa virus diseae on a nation-wide scale.To choose four or five isolates with special from one location to get the whole genome sequence,and at last we get 6 RNA1,31 RNA2,30 RNA3 from 111 isolates,combined with the data collected by the team in the previous three years,the molecular genetic structure of AMV population was analyzed.It was found that the population of alfalfa mosaic virus has formed a relatively stable population,and the pressure of MP protein selection of the virus has increased with the years,which means that the MP is constantly adapting to the environment including the host and natural environment for evolution.Combination with the published data from Gen Bank,the phylogenetic analysis of of the AMV population was analyzed,the diversity of AMV of Chinese population is rich.In the found that the diversity of isolates in China is rich.In the phylogenetic trees of RNA1 and RNA2,the isolates from Lanzhou,Gansu had the clssic geographyic characteristics.The isolates from Cangzhou,Hebei were also had the same traits in RNA2 ML tree.Furthermore,the isolates from Sapin and Wulan,Qinghai were clustered in RNA1,RNA2 and RNA1+2+3 ML trees.The Italian isolate was grouped with the XIANGGELILA isolate in RNA2 ML tree,which means the isolates from western resion of China and European had the same evolutionary ancestor.We analyzed the molecular genetic structure of CP gene of AMV population of China,Iran,Spain and the world by the BEAST and Ba TS softwares.Based on the analysis of AMV-CP,The disease occoured in Spain is earlier than China and Irin,the populations in China and Spain groups respectively,the difference is existed.In the world,there are very significant differences between the populations of different countries.But at the species or genus level,the populations do not show significant differences.This is the first time for the analysis of comparison for the population of AMV-mp from different countries in the worldIn terms of the infective clone population research of alfalfa mosaic virus,the pure c DNA clone virus population of AMV(RNA1: RNA2: RNA3)was used.Using the RT-PCR to get the RNA1,RNA2 and RNA3 from the c DNA clone plasmid,then noculated on the resistant varieties of Gannong 5 and sensitive varieties of Zhongmu 1 continuously.At present,it has been subcultured for 9 generations,and the positive rate of AMV of Zhongmu 1 increased from 76% to 92%.In the eighth generation,the positive rate of AMV of Gannong 5 increased from 0%(the first generation)to 72%(the eighth generation),and the disease symptoms were more and more abundant,which indicated that the pathogenicity and adaptability of alfalfa mosaic virus on different hosts increased gradually with the increase of generations.However,the significant difference of AMV positive rate between the two hosts reflects the difference of pathogenicity and population adaptability of AMV to Zhongmu 1 and gannong 5,which lays a foundation for further revealing the genetic variation sites of AMV in different resistant and sensitive varieties.