| The occurrence of tomato spotted wilt virus(TSWV)will have an unpredictable impact on the production of tomato and other economic crops in Ningxia.At present,the development of Sw-5 marker is less at home and abroad,and there is no report about the identification of the virus in Ningxia.In this study,14 samples of diseased tomato leaves collected from Yinchuan city were identified by RT-PCR using the national standard detection primers of TSWV.The target band of TSWV-N gene was cloned and sequenced.Multiple sequence alignment and phylogenetic tree analysis were carried out.TSWV antibody was further detected by Western blot to determine whether the tomato samples were infected by TSWV.Used the method of friction,spray and mechanical inoculation,we identified the positive leaf samples of 62386-7-0,screened the best method to identify the disease resistance,and used the disease resistant materials D61358P-0,susceptible materials 62386-7-0 and the F2 population constructed by D61358P-0×62386-7-0 was used for disease resistance identification.The SSR molecular markers closely linked to TSWV resistance gene were screened out by extracting DNA from parents and individual plants of population.The genetic distance was calculated and the genetic map was constructed.The selected SSR markers were used to detect 34 tomato germplasm resources,and the reliability of SSR markers was verified by the results of field disease resistance identification.The results show that:(1)The sequence similarity with TSWV-N ranged from 97.45%to 99.49%.It was closely related to the gene sequences of viruses infecting tomato in Yunnan,lettuce in China,iris in China and capsicum in Chongqing,It was also related to viruses infecting Shandong,Heilongjiang and Chongqing The virus gene sequences in Beijing and other places are relatively distant.The results showed that the protein of tswv-n gene was similar to the expected size in 8 samples.The results of molecular identification and protein detection showed that the diseased tomato leaves collected in Yinchuan were infected by TSWV.(2)The incidence rate and incidence index of mechanical method,friction method and spray method were significant difference.Among them,mechanical method had the best effect,the incidence rate was 82.41%,the disease index was 63.23,the spray method was the worst,the incidence rate was 31.46%,the disease index was 25.87,and the friction method was between two.(3)There were significant differences in resistance and susceptibility between male and female parents.The identification of disease resistance of female parents showed resistance,while that of male parents showed susceptibility;Among 409 F2 populations,11 were dead,297 were resistant and 101 were susceptible.The segregation ratio of resistance and susceptibility was 3:1,which was consistent with single gene dominant inheritance.(4)Among the 70 pairs of SSR primers,10 pairs of primers with clear bands were amplified from both parents,which were ND146,ND486,ND488,ND489,ND492,ND513,ND526,ND529,ND534 and ND538,respectively.Three pairs of SSR primers,ND146,ND489 and ND 492,were amplified from the susceptibility pool.The genetic distances of three pairs of markers ND146,ND492 and ND489 were 0.98 cM,4.56 cM and 9.84 cM,respectively.(5)Among the three SSR markers,the coincidence degree of ND146 molecular identification and field resistance identification was 82.35%,and that of ND489 and ND492 was 88.23%.The above results provide a theoretical basis for the identification of TSWV,the identification and screening of disease resistance of germplasm resources,and the implementation of fine variety breeding in Ningxia.The 3 pairs of SSR markers closely linked with TSWV resistance genes can be used in Tomato Molecular assisted breeding. |
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