Genetic Analysis And Gene Mapping Of Tobacco Branchy Mutants

Plant branch development has a very important position in plant morphology,Molecular biology research on plant branch development is mainly based on mutants that have variation in branch morphology.Most of the plants currently studied on branching are tomato,pea,maize,rice and Arabidopsis thaliana,and a number of mutants associated with stem branching structures have been identified in these plants,which laid a foundation for the molecular mechanism of plant branch development.In this study,two multi-branchy mutants,mbd1（more branch and dwarf 1）and mbd2（more branch and dwarf 2）were identified and genetically analyzed.The main results are as follows:（1）Compared with Honghuadajinyuan（HD）,there was no obvious phenotypic difference between the wild-type HD and the mbd1 mutant before the fourth week after transplanting.After that,axillary buds could be observed to appear initially on the adaxial sides of leaf base in both HD and mbd1.At the sixth week after transplanting,mbd1,however,showed several significantly longer branches developed from the axillary buds on the main stem base than HD.At the eighth week after transplanting,the difference of branch length between HD and mbd1 was further increased.Nevertheless,there was no significant difference between HD and mbd1 in the number of buds and/or branches during this period.After that,the branches of HD seemed to be dormant,whereas that of mbd1 continued to grow up.However,compared with HD,the elongation of the main stem of mbd1 began to slow down,resulting in a dwarf phenotype at the flowering stage.With the continuous development,the mbd1 mutant eventually formed a bushy appearance after maturity.（2）Compared with CuiBi No.1（CB-1）,the main features of mbd2 mutant:many axillary buds on the adaxial sides of leaf base,axillary buds growing strong,the rapid growth of axillary buds after flowering and showing a clustered appearance ultimately.（3）F₁,F_1’,F₂,F_2’,BC₁F₁and BC₁F_1′were constructed by wild-type（HD and G3）and mbd1.Genetic analysis showed that F₁ and F_1’were normal branching phenotype,indicated that the mutant phenotype of mbd1was controlled by a sigle recessive gene.The segregation ratio between mutants and normal plants was 3:1 in F₂ and F_2’,and the segregation ratio between mutant and normal plants was 1:1 in BC₁F₁and BC₁F_1′,indicated that the mutant phenotype is controlled by a sigle recessive gene.（4）The genetic populations of F₁,RF₁,F₂,RF₂,BC₁F₁ and RBC₁F₁ were constructed by CB-1 and mbd2.The genetic analysis showed that F₁ and RF₁ were normal branching phenotype,indicated that the mutant phenotype was controlled by a sigle recessive gene.The segreation ratio of mutants and normal plants in was 3:1 in F₂ and RF₂,the segreation ratio of mutant and normal plants was 1:1 in BC₁F₁ and R BC₁F₁,indicated that the mutant phenotype was controlled by a single recessive gene.（5）Given the similar mutant phenotypes and genetic mechanisms,it was probable that the mbd1 and mbd2 mutant contained the same mutant alleles.To check the hypothesis,we performed an allelism test by reciprocally crossing mbd1 with mbd2.The results showed that both F₁ displayed the similar phenotypes to mbd1 and mbd2 that were not observed segregated in their corresponding F₂populations,which indicated that the same recessive nuclear gene controlled the mutant phenotypes of both mbd1 and mbd2 mutants.（6）The BC₁F₁ population with segregation ratio of 1:1 for much-branched plants and normal plants was utilized as mapping population.Using SSR markers,mbd1 was lacated between marker PT52548 and PT50934,with genetic distances of 5.2 cM and 3.9 cM,respectively.（7）A total of five non-synonymous mutations were detected by genomic re-sequencing using BC₁F₁backcross population which was derived from G3 and mbd1 mutant as well as the two parent lines.Three of the five mutations were located on chromosome 18,one on chromosome 24,and one on scaffold.According to our mapping results using SSR markers as well as the SNP index given by re-sequcing（index≥0.9）,mutation for mbd1 might be located on chromosome 18.What’s more,the five candidates from mbd1 were sequenced and only one base mutation was identified in one candidate gene.This gene was sequenced for each BC₁F₁ plants and multi-branched plants with no-mutations in this gene were discovered,indicating that this gene was unlikely to be the mbd1 gene.（8）Based on bioinformatics analysis,the HD-zip III genes of cultivated tobacco genome TN90 were screed and selected,the research provided the theory foundation for deep analysis of NtHD-zip III gene foundation.