| The powdery mildew could affect the yield and quality of tobacco seriously.At present,in order to control tobacco powdery mildew,using fungicides remains to be the general method although it may have a serious impact on the environment,and potential risk of exceeding pesticide residues.Therefore,breeding powdery mildew resistant varieties on the basis of resistance genes identification is an safe,economical and effective method for tobacco powdery mildew control.In this study,nine powdery mildew resistant materials were identified from different types of tobacco germplasm resources.Two important resistance sources were chosen for genetic analysis and function verification.We clarified that NtMLO genes played the pivotal role in the powdery mildew resistance materials,and analyzed the mechanism of mlo mediated resistance.The main results are as follows:1.The optimized inoculation and evaluation method for tobacco powdery mildew was established,not only suitable for tobacco seedling but also for in vitro leaf and the whole plant.Using this method,321 tobacco germplasm resources,covering different tobacco types such as flue-cured tobacco,sun cured tobacco,air cured tobacco,burley tobacco and cigar were inoculated with powdery mildew and surveyed 15 days later.Among them,9 resistant materials were founded,including flue-cured tobacco and sun cured tobacco.That provides not only a good parent material for disease resistance breeding,but also essential information of the resistant characteristics to powdery mildew in tobacco germplasm resources.2.Sequence analysis of Taiyan 7 and Tang Peng showed that both ntmlo1 and ntmlo2 mutations could be detected.Tang Peng and Taiyan 7 contained the same ntmlo1 mutant sequence that the 3’ alternative splicing site of the seventh intron was changed because of the last 2 nucleotides AG were mutate to TA.This kind of mutant could lead to 6 chimeric splicing variants.Comparing to wild type,the forth and fifth nucleotides AG were deleted in 5’ terminus of the sixth intron in ntmlo2 of Taiyan 7mutant.But there was a 556 bp fragment,containing the 3 ’end of the fifth intron,the whole exon 6 and parts of 5’ terminus of the sixth intron,inserted into the sixth intron in ntmlo2 of Tang Peng mutant.This kind of mutant in Tang Peng could lead to the transcript containing a complete repetition of exon 6(89bp)and the translated amino acid sequence aberrant after 198 amino acid and being terminated at 228 amino acid.Molecular markers were developed according to the mutation sequence and verified by the segregation population.It was found that the markers were co-separated from the disease resistance phenotype.3.A total of 29 candidate sequences of NtMLO family in tobacco were identified by whole genome sequence analysis.Through bioinformatics analysis,we found that tobacco NtMLO protein contains 479 ～ 605 amino acids,and 5 ～ 7 transmembrane domains.Tissue expression analysis showed that most genes were expressed in root,stem,leave and flower.The expression of NtMLO1,NtMLO4,NtMLO13 and NtMLO16 genes will be induced by powdery mildew,while the expression of NtMLO7 and NtMLO12 genes will be inhibited.We also found that PEG,ethylene,GA,SA,JA and ABA can induce or inhibit the expression of NtMLO3,NtMLO4,NtMLO7,NtMLO9,NtMLO12,NtMLO13 and NtMLO16 to varying degrees.The function of NtMLO in tobacco powdery mildew susceptibility was verified by Tang Peng with NtMLO1 over-expression and K326 and Guiyan11 with the ntmlo1ntmlo2 double mutants created by CRISPR/Cas9.4.The mechanism of mlo-mediated powdery mildew resistance was clarified by omics analysis.It was found that the pathway related to cell wall synthesis played a key role in the process of disease resistance.Over-expression of NtXTH1 and NtERF1 in mlo-mediated disease resistant materials can partially restore the susceptibility to powdery mildew,but knock out NtERF1 gene in susceptible materials can not obtain disease resistance.Yeast single hybridization confirmed the interactions between NtXTH1 and transcription factors NtERF1.It suggested that cell wall strength could increase substantially when NtXTH1 was down-regulated significantly as a result of which transcription factor NtERF1 decreased sharply.That means NtXTH1 and NtERF1 transcription factor could play a novel important role in mlo-mediated powdery mildew resistance.5.The allele-specific molecular markers were utilized to assist the oriented genetic improvement of K326 and Yunyan 87.The created tobacco new lines had no significant changes in agronomic,yield,economic and quality traits except that the resistance against powdery mildew was stronger than the original varieties.New powdery mildew resistant lines not only can be directly applied in production,used as intermediate materials for pyramiding breeding,but also can be used to evaluate the induction rate of haploid induction lines. |
