| Alfalfa is one of the most significant pasture crops in the worldwide,however,the occurrence of Alfalfa virus disease has caused severe losses to the production of alfalfa.It is of great significance for the prevention and control of Alfalfa virus diseases by determining the types of Alfalfa virus diseases and screening disease resistant varieties.The second-generation sequencing technology has broken through the limitations of traditional virus detection methods which cannot detect unknown viruses,and has become one of the frontier technologies for viral detection.Based on the field investigation on the incidence of viral diseases in 30 alfalfa varieties and by using the comprehensively compare the resistance of different alfalfa,we hope to provide scientific evidence for the development and utilization of alfalfa varieties germplasm resources.To identify the types of viral diseases in 30 varieties of Alfalfa in Henan,we used small RNA deep sequencing technology.Alfalfa mosaic virus(AMV)was detected in 30 varieties of Alfalfa by the contigs splicing and assembling.To further exploring the molecular mechanism of the AMV resistance in alfalfa invasion,we used the deep analysis method to research the small RNAs about AMV.The experiment is divided into three main parts as follows:Exp.1: Study on the field resistance among 30 alfalfa varieties to viruses.Experimental results show:(1)The field incidence of all alfalfa varieties was more than 94%,and the range of disease index ranged from 31.53 to 55.50.(2)There were 1 resistant varieties,6 intermediate varieties,and 23 susceptible varieties,accounting of 3.33%,20.00%,76.67% of the tested germplasms of 30 alfalfa varieties,respectively.(3)The cluster analysis indicated that Sheshou No.2 had a far relationship with all other alfalfa varieties,respectively.Exp.2: Identification of AMV by deep sequencing of small RNA.Experimental results show:(1)The small RNA deep sequencing technology sequences was deployed to assemble the AMV sequence,which showed this new sequence was 7217-nts and account for 87.23% of AMV total length(8274-nts).(2)BLASTn analysis showed an overall nucleotide identify of AMV.Primers were designed based on AMV-derived si RNA,the position of contigs located on the AMV genome as described above,and corresponding reference genome sequences.PCR amplification of AMV,tapping recovery and clone sequencing.A total of 5344 bp fragment was amplified,and the homology with the AMV genome was as high as 96%.Among them,the homology with the coat protein gene of AMV was 99%.(3)The phylogenetic tree of AMV-CP genomes was constructed by MEGA.It was found that the AMV isolate obtained from this study had close relationship with AMV isolates from Korea,and had far relationship with AMV isolates from Iran.Exp.3: Deep analysis of the small RNA derived from AMV.Experimental results show:(1)It was different between the polarity distribution of si RNAs derived from different genomes of AMV,and the number of si RNAs from the sense strands of the three AMV genomes was higher than that from the antisense strands.(2)The length distribution of si RNA from different genomes of AMV tends to be consistent,with si RNAs of 21 nt and 22 nt in length occupying the major part.(3)There was different in the 5' terminal nucleotide preference of si RNAs derived from different genomes of AMV.Among them,AMV-derived si RNAs starting with “U” or “C” are more numerous than those beginning with “A” or “G”,and This rule is also observed in AMV-derived si RNAs of different lengths.(4)There was different in the distribution of hotspots of si RNA from different genomes of AMV.The hotspots of AMV-RNA1-derived si RNA are mainly concentrated in the AMV-RNA1 genome from 600 to 800 positions.the hotspots of AMV-RNA2-derived si RNAs are mainly concentrated in the The location of AMV-RNA2 genome is from 1190 to 1370.The hot spot region of AMV-RNA3-derived si RNA is mainly located in the AMV-RNA3 genome at positions 1241 to 1580. |
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