Genetic Analysis And Gene Mapping Of Tobacco Space Mutant Leaf Trait

Tobacco is an important economic crop in China.The selection of tobacco varieties with excellent traits is the key to producing high quality tobacco leaves.However,due to the reason that tobacco was introduced from foreign countries,the lack of germplasm resources is the bottleneck of tobacco breeding in China.The new germplasm of innovative tobacco is the urgent task of tobacco breeding.Aerospace breeding,which combines aerospace technology with conventional breeding,is one of the new ways of germplasm innovation.In this study,the tobacco NC89 space mutant returned by the"Practice 8"breeding satellite was used as the experimental material,and the agronomic traits and the quality traits of the roasted tobacco were analyzed.The whole genome resequencing was used to reveal the difference between the mutant and the wild type genome.The F₂ population was constructed using aerospace mutants to focus on the genetic analysis and gene mapping of leaf-shaped mutant traits,and combining with resequencing data to predict candidate genes and analysis in the localization interval.The findings are as follow:1.The tobacco mutant material NC89-M was created by aerospace mutagenesis.Compared with the wild type NC89,the mutant leaf shape was broadly elliptical,the leaf surface was wrinkled,and the leaf margin was serrated.The F₂ population was constructed by using NC89-M and tobacco new strain2014-168.The results of genetic analysis indicated that the NC89-M leaf-shaped mutant trait was controlled by a dominant major gene.The SSR marker PT60795 was closely linked with it by BSA method,and the genetic distance was 2.9 cM.2.The whole genome re-sequencing of tobacco mutant material between aerospace mutant NC89-M and wild-type NC89 was performed,the sequencing depth was 30×,and various types of variation were detected and annotated.In the NC89-M,1876219 SNPs,402011 Indel,42699 SV and290218 CNV were detected.In the NC89,1848013 SNPs,398922 Indel,41969 SV and 290939 CNV were detected.A total of 271655 SNPs were obtained from NC89-M and NC89,distributed over 8378genes,and 23450 Indel causing 2156 gene mutant.The KEGG annotation of the variant gene indicated that the number of gene mutations was the highest in both metabolic pathways and secondary metabolite synthesis.16 homogeneous SNPs were randomly selected from the resequencing data,14 of which were correctly verified,and the correct rate was 87.5%.3.According to the resequencing data,new Indel markers were developed between NC89-M and tobacco strain 2014-168,and the mutated leaf gene nc89m was finally mapped between the SSR marker PT60795 and the Indel5 marker with a genetic distance of 2.9 cM and 4.4 cM,respectively.The gene Nitab4.5₀000008g0880.1,which was highly expressing in the leaves,was selected as a candidate gene in the localization interval.The NCBI Blastp alignment of this gene indicated that it is the glycosyltransferase SQD2 gene,so call the gene NtSQD2.Gene sequence amplification indicated that the 1294th nucleotide of the coding region in the gene was changed from adenine to cytosine,and the encoded amino acid was mutated from tyrosine?Tyr?to aspartic acid?Asp?in the NC89-M.The qRT-PCR results showed that the expression of NtSQD2 gene in NC89-M leaf tissue was significantly up-regulated compared with NC89;bioinformatics analysis indicated that NtSQD2 encoded protein contained a highly conserved glycosyltransferase domain.This study laid the foundation for further analysis of the mechanism of space mutagenesis,the cloning and functional verification of key genes regulating leaf traits.