Identification Of Co-dominant Molecular Markers Linked To TMV Resistance Gene N And It's Application In Flue-cured Tobacco Breeding

Tobacco mosaic,a frequently occurring world-wide disease in tobacco production,has posted serious threat and caused great losses to the tobacco industry.To date,there has been no effective measure to control the tobacco mosaic disease,while development of tobacco varieties with resistance to Tobacco mosaic virus?Tobacco Mosaic Virus,TMV?which is the main pathogen of tobacco mosaic,has been believed to be the most economical and effective way.It has been reported that N gene is the major tobacco gene offering TMV resistance.In this study,we took Coker176?a flue-cured tobacco variety with high resistance to TMV?,Y3?a flue-cured tobacco variety susceptible to TMV?and their BC₁F₁?Y3: backcross parent?population as experimental materials.In order to obtain a co-dominant marker tightly linked to tobacco N gene for marker-assisted selection?MAS?of N gene in the population,18764 pairs of SSR primers were used to screen for simple sequence repeat?SSR?markers by bulked segregation analysis?BSA?.The main results are as follows:1)Identification of polymorphism for the tobacco genomic SSR markers: PCR amplification was performed with 18764 pairs of primers for five materials?Y3,Coker176,F1,susceptible pool and resistant pool?.The results showed that,among the 18570 pairs of SSR primers?99%?which worked well for the five experimental materials,only 396 SSR markers were generated to be co-dominant marker which are not only present in both two parents but also in F1.In agreement with previously reported studies that narrow genetic basis,close genetic relationship,and low genetic diversity exist between the varieties of the N.tabacum?especially flue-cured tobacco grown in large area?,this result also indicated a very low polymorphism rate of 2.11%?396/18764?SSR markers between Y3 and Coker176.2)Acquisition of SSR marker linked to TMV resistance: The genetic linkage analysis of 396 polymorphic SSR markers and the target gene?N?was carried out by bulked segregation analysis?BSA?.The results showed that the band,closely linked to TMV resistance gene N,could only be amplified by one pairs of SSR primers?TM508-007?.The genotype of the linked marker?TM508-007?in 123 BC1F1 plants was analyzed and the linkage distance between the marker and TMV resistance gene N was calculated by using software Joinmap 4.0 to be about 0.41 cM.3)Verification of resistance linked marker: The validity of the TMV resistance linkage marker?TM508-007?obtained from this study was conducted using 16 TMV flue-cured tobacco varieties from different sources of resistance and different genetic background.The genotyping analysis of 16 flue-cured tobacco cultivars were not only consistent with the phenotype anlysis of TMV resistance in the field,but also can be used to clearly distinguish the homozygous resistance,the hybrid resistance and the resistance of different sources.These results showed that the marker is closely linked to the target gene N and an ideal co-dominant marker with the characteristics of stability,reliability and high efficiency.This co-dominant SSR marker enriches the number and types of anti-TMV molecular markers of tobacco,and can be used for MAS of tobacco resistance to TMV.4)Application of resistance linked markers: 18 excellent disease-resistant plants with homozygous disease resistance and good field performance were obtained by MAS of marker TM508-007 and comprehensive evaluation of the main agronomic characters in field.The 18 resistant plants can be further used for disease resistance tobacco breeding.