Genome-Wide Association Study And The Candidate Genes Prediction Of The Nicotine Content In Tobacco

Nicotine is one of the most important quality traits of tobacco.To analyze the mechanism of nicotine content and screen the nicotine content of tobacco varieties will provide important guidance for tobacco quality modif ication.However,directly screening of nicotine content from varieties consume huge manpower,material and financial resources,and is time consuming and low inefficiency for taking advantage of tobacco germplasm resources.Screening by molecular markers can effectively shorten the screen time,save costs,and greatly improve work efficiency.In recent years,several nicotine related molecular markers have been identified,but they are rare used in the selection of tobacco germplasm resources for the low density and accuracy.With the rapid development of the high-throughput sequencing technology,RAD sequencing technology has been widely used because of its advantages such as uniform genome coverage,low cost,short cycle and so on.And base on SNP loci Genome wide association analysis has been rapidly developed as an effective tool for the study of complex quantitative genetic traits in plants.In this study,RAD sequencing of 219 flue-cured tobacco varieties and whole genome sequencing of 29 Flue-cured Tobacco Varieties was carried out.SNP loci,which were signif icantly correlated with nicotine content variation and nicotine synthesis key gene expression were explored by Genome wide association analysis.Further,the prediction of corresponding candidate genes and exploring of functional tag were developed.In general,this study will give useful guidance to identification of the nicotine related molecular markers and breeding of high nicotine varieties.Results are as follows:1.RAD sequencing of 219 flue-cured tobacco varieties was carried out,and the data of the 384,904 high quality SNP loci were obtained.Based on the phenotypic identification of nicotine content in flue-cured tobacco leaves in 8 years/sites,with the method of whole genome association analysis,on the basis of the signif icance threshold,2 SNP loci were found to be significantly associated with nicotine content,which were located at 69912063 bp on chromosome 1 and at 81115794 bp on chromosome 2.Based on the tobacco genome database,we predicted 2 candidate genes,Ntab0691660 and Ntab0574860,respectively.The candidate gene Ntab0691660 is homologous to the ATCYS6 gene in Arabidopsis,and it is speculated that Ntab0691660 may be involved in the regulation of nicotine biosynthesis by regulating the pathway of biological stress resistance.The candidate gene Ntab0574860 is homologous to the OSJMJ706 gene in Rice,and Ntab0574860 may be related to the gene expression regulation of the nicotine biosynthesis key enzyme putrescine N-methyltransferase.The allele specific PCR?AS-PCR?markers were designed for the SNP loci detected by association analys is.1 available marker AP014 was screened in C1₆9912063.SNP site C2₈1134669 was close to the association site C2₈1115794,and 2 AS-PCR markers?AP068 and AP072?with clear bands were screened from the site.The study provides a useful marker and gene resource for the excavation of the high nicotine genotypes and the molecular marker assisted selection of new varieties of nicotine2.Whole genome sequencing of 29 flue-cured tobacco varieties was c arried out,and based on the variation of the genome sequence,SNP and Indel mutations of 4 PMT family genes in common tobacco species were analyzed.A missense mutation in the SNP?C23₄9502707?was found in the seventy-fifth nucleotide positions in the exons of the gene Ntab0153810?NtPMT2?.Compared with the reference genome,the SNP was mutated from T to G,in some varieties.As a result,the translation of amino acid sequence changed from histidine to glutamine,and the amino ac id properties changed fr om basic amino acid to amide amino acid.The results of variance analysis showed that the average content of nicotine in T genotype was 14.7% higher than that of G genotype.T genotype is an excellent variant of high nicotine.For this site,functional marker transformation was carried out,and 1 pairs of stable and clear bands were screened for AP058.The present study provides a useful marker and gene resource for molecular marker assisted selection of high nicotine content.3.Total 219 flue-cured tobacco varieties were analysis by the nicotine synthesis key gene PMT,QPT expression level.According to the high quality SNP data obtained by RAD sequencing,the result of Genome-wide association analysis showed that?1?2 SNP loci associated with PMT expression were detected on the threshold of significance,located at 76585016 bp on chromosome 2 and 150848920 bp on chromosome 5,respectively.?2?8 SNP loci associated with QPT expression were detected on the threshold of signif icance,located at 2177629 bp and 2177631 bp on chromosome 2,150426307 bp on chromosome 4,15845870 bp,107612287bp,107612311 bp,107612331bp and 107612344 bp on chromosome 9.In this study,hydroponic culture was used to cultivate tobacco seedlings in the artificial climate chamber to ensure a stable consistent cultivate environment.And the expression level of the nicotine synthesis key gene was used as the phenotypic trait to reduce the time cost of molecular markers exploring,the result provided a new way for the screening of molecular markers.