Establishment And Application Of Colorimetric And Rapid Detection Of SMV-SC3 Besed On Loop-mediated Isothermal Amplification (LAMP)

Soybean mosaic virus is the most common and serious disease in soybean diseases.Soybean infected with soybean mosaic virus not only affects soybean yield,but also causes soybean quality decline.Cultivation of SMV resistant soybean varieties and early detection of soybean mosaic virus pathogens are very important to improve soybean yield and ensure soybean quality.SC3 strain is a weak strain,which has no obvious symptoms at the early stage of infection in soybean leaves,but it can seriously affect soybean yield.Shanxi is one of the main soybean production areas.And it is important to study the resistance of soybean germplasm resources to Mosaic virus disease and to establish a simple,rapid,accurate,economical and develop a specific SMV-SC3 identification method forsoybean disease resistance breeding and soybean production in future.In order to investigate the infection of soybean Mosaic virus in soybean resources suitable for planting in Shanxi,238 soybean germplasm resources were planted in experimental field of Shanxi Agricultural University in 2020.The infection of plants and seeds of 238 soybean germplasm resources were investigated,The NIb and CP genes were cloned,sequenced and phylogenetic tree analysis.According to the polymorphism of CP genomes of different soybean mosaic virus strains,LAMP primer set for SMV-SC3 were designed.Through screening primer groups,optimizing reaction conditions,specificity and sensitivity,a visual method for detecting SMV-SC3 with high specificity,high sensitivity,simple operation and economy was established.Finally,26 soybean materials were visually detected by this method.1.Through the investigation and analysis of soybean plant of 238 soybean germplasm resources,four typical symptoms of soybean Mosaic virus were found in the field,among which 47 soybean materials showed shrinkage and mosaic symptoms,126 showed shrinkage,48 showed mosaic symptoms,and 7 showed terminal withered symptoms,10 soybean materials was normal.Through investigating appearance of seeds in 238 soybean germplasm resources,24 soybean seed coats were found to have typical soybean Mosaic virus spots among 117 yellow seed coats and 3 green seed coats,.Through the investigation and analysis of soybean plant,10 soybean materials resistant to SMV were screened,and they were SZ-97,SZ-1022,Jindou 42,Zhonghuang 40,PB2437,Fendou 92,Fendou 93,Fendou 103,Fendou 105,and Mandou 14.These materials can be used as disease-resistant soybean breeding materials in the future.2.The SMV genomic sequence was cloned from the leaves of SMV susceptible plants.By sequencing,alignment and phylogenetic tree analysis,SZ-319,Fendou 78,SZ-1042,SZ-869 and SZ-1058 were found to be clustered in the same classwith SMV-SC3 strains.SZ-834 and Yuandou 1 were grouped with SMV-SC21 strain.3 According to the sequence differences between SMV-SC3 and other strains of soybean Mosaic virus,two specific LAMP primer set were designed and found that primer set 2 could effectively conduct LAMP reaction of SMV-SC3.The system of LAMP reaction was optimized,and 64℃ reaction temperature and 0.4 mol/L betaine concentration were found to be the optimal reaction system.The specificity test showed that primer set 2 could specifically detect SMV-SC3 virus.The sensitivity of LAMP for detection of SMV-SC3 virus was 0.01 ng/μL,100 times higher than that of conventional PCR.4 The established SMV-SC3 LAMP reaction platform was used to detect SMV-SC3 in the leaves of Nannong 1138-2 at different times after inoculation with SMV-SC3.It was found that SMV-SC3 virus could be detected 10 days after inoculation,but Mosaic symptoms could not be seen until 15 days after inoculation with naked eyes,indicating that the SMV-SC3 LAMP developed in this study could be used for early detection of SMV-SC3 strain.Using the established method of LAMP identification of SMV-SC3 virus,the plant leaves a of 26 soybean germplasm resources were detected.and found that Roundbean 1,SZ-834,Fendou 92 and Handou 14 were not infected by SMV-SC3 strains,and t 22 materials were all infected by SMV-SC3 strains.In conclusion,a visual method with high specificity,high sensitivity,simple operation and economy for the detection of SMV-SC3 by LAMP were established.Four soybean materials resistant to SMV-SC3 were screened.