Screening Of Tobacco Germplasm Resourses Resisted Tobacco Mosaic Virus (TMV)

In this paper, the genetic diversity and genetic relationship for three varieties of tobacco geramplasms by ISSR molecular marker were researched. In order to lay a foundation for selecting tobacco geramplasms resisted disease, three kinds of tabacco geramplasms resistance against TMV were identified in both molecular identification and field identification. Finally, The temporal and spatial expression of gene (cn) resistance against TMV was performed to analyze taking three different resistance of tobacco varieties, which were susceptible cultivar Youmeimimu, resistant cultivar LongyanC2, highly resistant cultivar Yongding400 as experiment materials by semi-quantatitive RT-PCR for the sake for providing the theory basis for early diagnosis and prevention of tobacco mosaic virus.The primary research results were as followed.1、The investigation of the genetic diversity and genetic relationships of three kinds of tobacco geramplasmsThe 24 polymorphism primers were selected from 100 ISSR primers.141 polymorphism bands were attained from 192 DNA bands, and the average ratio of polymorphism was 73.4 percent. The outcome of ISSR statistical data for three kinds of tobacco geramplasms by DPS was also attained. The genetic similarity for three kinds of tobacco geramplasms ranged from 0.1258 to 0.5294. Thus, the results illustrated that genetic diversity was relatively plentiful and existed obviously genetic difference, so they had relatively wide genetic basis. While in the process of Cluster Analysis, some tobacco geramplasms had relatively close genetic relationship which would gather into a large class. The same kind of tobacco geramplasms existed obviously genetic difference was not necessarily gotten together. And the tobacco geramplasms, which would also occur the crossing cluster phenomenon, had close genetic relationships.2、The investigation of three kinds of tobacco geramplasms resistance to TMVThe results of molecular identification were as followed. A pair of sepicific primers were designed according to the reported gene (cn) resistance to TMV sequence in GenBank. And the genomic DNA was amplified from three kinds of tobacco geramplasms by PCR, so 36 resistant bands were detected. Then, PCR products were recycled and purified in order to conduct sequence analysis, the result confirmed that the conservative nucleotide sequence of gene was 88% homologous to other genes (cn) resisted TMV.The results of field identification were as followed. By inoculated three kinds healthy plants with TMV artificially in field, the results showed that each tobacco geramplasms had its own defence performance. There were 4 highly resistant tobacco geramplasms,3 highly susceptible tobacco geramplasms,25 resistant tobacco geramplasms,14 susceptible tobacco geramplasms among 46 tobacco geramplasms.After the comparison and analysis between the results of molecular identification and field identification,28 of 46 tobacco geramplasms were consistent between molecular identification and field identification, its rate was 60.9 percent. In addition, it had never been undertaken the investigation of 24 tobacco geramplasms resistance to TMV by our forefathers. Moreover, the results of molecular and field identification to 16 tobacco geramplasms was consistent.3、The investigation of temporal and spatial expression of gene (cn) resistance to TMVTotal RNA was extracted by the improved method of TRIzol. Two pairs of primers were designed in accordance with the reported housing gene (actin)of tobacco(actin as internal frame) and gene (cn) resisted against TMV sequences of GenBank to conduct the semi-quantatitive RT-PCR. After inoculated them with TMV, three kinds of tobacco varieties of gene (cn) resisted against TMV had different expression in different time The susceptible cultivar Youmeimimu (A9) began to develop mosaic symptoms on the leaves of tobacco in 10d, the expression of resistant TMV gene (cn) was highest. The resistant cultivar LongyanC2 (C17) began to develop mosaic symptoms on the leaves of tobacco in 2d, the expression of resistant TMV gene (cn) was highest. The highly resistant cultivar Yongding400 (C5) began to develop mosaic symptoms on the leaves of tobacco in 10d, the expression of resistant TMV gene (cn) was highest.The genetic diversity, genetic relationships, the identification of resistant TMV and the temporal and spatial expression of gene (cn) of three kinds of tobacco geramplasms had been studied in this paper. It is greatly profound influence to identify and select tobacco resisted against TMV. The results could lay a foundation for breeding resisted TMV varieties.