| This experiment aims at exploring the genetic rule of tobacco’s resistance to black shank,providing tobacco breeding against black shank with theoretical basis,rendering molecular assisted selection with effective SSR molecular marker and enhancing the efficiency of tobacco breeding for disease resistance.The experimenter carried out cross-breeding on black shank disease resistant variety Yanyan 97 and self-fertility hyperkalemia black shank disease induced variety GK2 as parents,identified the resistance of the parents,hybrid F1,F2,and BC1 population to black shank disease and analyzed their genetic laws according to the identification results.Experimenters collected each generation of flue-cured tobacco samples to extract DNA,analyzed with SSR molecular marker and got access to SSR molecular markers linked with the anti-black shank disease gene.（1）16 strains applied with mycobacterium inoculation method at field got invasion at the incidence rate of 80%;20 strains treated with potting got invasion at the incidence rate of100%.18 strains applied with traumatic inoculation method at field got invasion at the incidence rate of 90%;19 strains treated with potting got invasion at the incidence rate of95%.Chi-square test results showed that different cultivation methods and different inoculation methods had the consistent effect.（2）The disease-resistant parent Yanyan 97 showed high resistance to No.0physiological race of tobacco black shank disease.The identification result of the individual plant was in line with the anti-infection ratio of 1R:0S;the susceptible parent GK2 was highly susceptible to tobacco black shank disease,and the identification result of the individual plant was in line with the anti-infection ratio of 0R:1S;hybrid F1 generation was moderately susceptible to tobacco black shank disease,and the identification result of the individual plant was in line with the anti-infection ratio of 0R:1S;F2 segregation group was moderately susceptible to tobacco black shank disease,and the identification result of the individual plant was in line with the anti-infection ratio of 1R:3S;4 combinations of BC1generation A:♂Yanyan97×♀（♀Yanyan 97×♀GK2）,B:♂GK2×♀（♀GK2×♀Yanyan97）,C:♀Yanyan 97×♀（♂GK2×♀Yanyan 97）and D:♂GK2×♀（♂Yanyan 97×♀GK2）all showed disease resistance,and the identification results of the individual plants were all in line with the anti-infection ratio of 1R:1S.（3）SSR-PCR was used to select the reaction system of pre and post primer 0.4μL,ddH2O13.5μL,dNTP 2μL,10×PCR buffer 2μL,rTaq enzyme 0.2μL and DNA template 1.5μL for amplification.The amplified product bands were clear and bright.（4）Among the 318 pairs of SSR primers,25 pairs showed polymorphism differences between the resistant and susceptible parents at the polymorphic rate of 7.86%.25 pairs of SSR primers with polymorphic differences between the parents were screened by F2generation for single plant to acquire the consistent polymorphic difference between the resistant individual and susceptible individual of SSR primer PT54175-216 with the parent.PT54175-216 is located on the short arm of chromosome of No.6 tobacco and linked to the black shank resistant gene of parent Yanyan 97.The PCR amplified band size is 216 bp.（5）PT54175-216 was tested in the F2 generation,with 40 strains resistant and 98 strains susceptible.The segregation ratio of resistant-susceptible genotype is line with the expected ratio of 1R:3S,and X2c=0.30,P0.05=0.950.The identification results of mycobacterium inoculation method in artificial inoculation resistance are reliable and valid,so the method is suitable for identification of resistance to black shank disease in a large number of field tobacco plants;the genetic inheritance of Yanyan 97’s resistance to No.0 physiological race of tobacco black shank disease may be controlled by a pair of recessive genes;SSR marker PT54175-216 presents stable polymorphism differences between parental and resistant-susceptible separated individuals with linked inheritance with resistance genes. |
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