Molecular Population Genetics Of Alfalfa Mosaic Virus In China

Alfalfa is the most important legume forage in the world.It is known as"the king of pasture".At present,alfalfa planting area ranks the first among all kinds of pasture in China,ranking the second in the world.Especially in recent years,vigorously developing the alfalfa industry is a sunrise industry that is in line with China’s agricultural development strategy,gaining ecological and social benefits.However,with the continuous enlargement of alfalfa planting area and continuous accumulation of continuous cropping and spreading mediators,alfalfa virus diseases are developing rapidly.The disease plants are seriously dwarfed,leaf shrinkage and yellowing,herbage protein content and herbage dry weight are greatly reduced,which seriously affect the yield and quality of herbage,and alfalfa virus disease has now become one of the most serious diseases.In this study,we aimed to identify the alfalfa viruses,and catch the information of the occurrence and distribution of the virus diseases,then get the molecular population genetics of the advantaged virus.In the research,405 alfalfa samples were collected from seventeen locations of twelve main alfalfa producing areas in China.It was identified whether it was infected by seven common plant viruses,such as Alfalfa mosaic virus(AMV),White clover mosaic virus(WCMV),Soybean mosaic virus(SMV),Tobacco mosaic virus(TMV),Cucumber mosaic virus(CMV),Bean yellow mosaic virus(BYMV)and Phytoplasma(16SrⅤ).Though the molecular method,the identification of every virus have get,the viruses infected these alfalfa samples were AMV,CPMV,TMV,BYMV and 16SrⅤ,the advantaged virus was AMV,the positive rate was 50.1%.The highest diversity of symptoms of AMV was reflected in Cangzhou,Hebei province.Four or five isolates with big differences in the variety of symptoms were selected for getting near whole genome sequence,at last,46 RNA1,50 RNA2 and 50 RNA3 were obtained.This was the first time for obtaining so many near whole genome sequences of AMV in the world.The phylogenetics of nature population of AMV was analyzed by MEGA7.0,DNAsp5.0 and RDP 4.Through the analysis of intraspecific polymorphism of nucleotide,it is found that the disease has formed a relatively stable population,which was easy to be prevalented.The selection pressure of the four proteins of AMV population were in a negative situation,which indicated there was no the new strain or new species come out,RdRP 1 had the highest d_N/d_S,then CP,MP and RNA were in the order.The phylogenetic tree of AMV’s RNA1,RNA2,RNA3,cp gene with partial fragments and concatenated sequence of RNA1+RNA2+RNA3 shew that the diversity of AMV population in China was rich,the separated clades with isolates from Langfang and the west provinces,reflected they had the regional limitation characteristics of evolution.Based on the phylogenetic tree of partial cp gene,there were 3 large groups of I,II and III,which indicated the new evolutionary clade existed in the China,the samples of Cangzhou,Hebei were dispersed in each group,which should be relevant with the diversities of symptoms.In the analysis of the phylogenetic tree of the 3 RNA concatenated,the isolate from the Ulan,Qinghai was clustered with the isolate from Spain in the bottom of the phylogenetic tree,which indicated they should be earlier evolutionary isolates,the origin of AMV virus in China will be well worth studying in the next step.Using RDP software,46 RNA1+RNA2+RNA3 concatenated sequences in China and 3foreign countries(Spain and Italy)were analyzed for recombination.It was found that there were 29 recombination events and 32 recombinants in the three concatenated RNA sequences.All of recombinants were from our country,some of them had paternal isolates from foreign countries in the recombinant process,all of these reflected the development of the alfalfa business in the world,it was possible to recombinate among the virus.Recombination analysis showed that RNA1,RNA2 and RNA3 were all hot regions,the hotspots focused on the boundaries of RNA2,which indicated the importance of RdRP2 function.In the analysis of protein selection pressure,it was also found that RdRP2 had the greatest selection pressure.These reflected AMV has carried out a large number of gene recombination for the adaption to the environment,and resulted in the increase of the non synonymous mutation and abundant variation.