| TILLING（Targeting Induced Local Lesions In Genomes）is a newly developed reverse geneticsapproach used to screen mutant population. TILLING has been widely adopted in various crop specieslike Arabidopsis, rice, wheat etc and a pool of functional genes has been successfully identified.Establishment of TILLING screening for tobacco is important tool to study functional genomics intobacco, clone the important genes controlling the various physiological and agronomic traits, study theunderlying mechanism controlling these traits, and to breed new tobacco varieties. In the present study,M2mutant population of cultivated tobacco species Zhong Yan100have been produced by0.6%EMStreatment and the segregated population were separated based on mutant types and phenotypic traits.Subsequently, we established an optimized system of LICOR-TILLING and CE-TILLING for screeningof mutant tobacco population. The primers were designed according to NtLS gene sequence, the NtLSmutants were screened and identified. The NtPAL4genotypes in M3population were also identified byusing the offer-mentioned methodology.This study has focused to cover the following areas to establish effective and reproducibleTILLING system in N.tabaccum.(1) TILLING screening of Nicotiana tabaccum M2mutants population produced by0.6%EMSand recovery of956lines and10,497mutants plants. Investigating956lines in M2population producedby EMS were carried out and and various abundant phenotypes were found including plant height, leafshape, plant type, and florescence. The segregation ratio of the traits in the mutant population revealedthat most of these mutations are recessive.(2) The appropriate volume of fluorescent labeled primers (forward and reverse) and CEL Ienzyme used in LICOR-TILLING system was optimized. The optimum concentration of fluorescentlabeled primers determined in the10μl PCR mix ranged from0.32to0.64μl (1μM) and1.12~1.6μl(1μM) for forward and reverse primers respectively. The concentration of CEL I enzyme and10×buffer optimized to cut the heteroduplex was0.6μl and1.5μl respectively. This leads to theestablishment and optimization of LICOR-TILLING technology system for N.tabaccum genomescreening.(3) The NtLS gene was analyzed in both N. tomentosiformis and N. sylvestris. Bioinformatics toolswere used to compare the genomic sequence of NtLS gene which showed that NtLS deleted in286bp-376bp region of N. sylvestris.Forward primer was designed and the PCR reaction was optimizedThe NtLS gene (GenBank: EU93558) was screened in M2population through Licor-TILLING system.A total of11mutant lines were identified and confirmed by sequencing in1,000M2lines induced by0.6%EMS.The estimated overall mutation frequency for the pilot assay resulted to be1/87Kb.TILLING screening system established in our study is faster,efficient and reproducible for mutant genesof known sequence and can play a vital role in tobacco functional genomics research in future.(4) The CE-TILLING technology was established in N.tabaccum by using M2-369screened byLICOR-TIILING as positive control. The CEL I enzyme digestion system was optimized and the concentration of CEL I enzyme and10×buffer used to cut the heteroduplex was0.2μl and0.6μlrespectively. To further optimize CE-Tilling system, PCR product with the restriction enzymeconcentration experiment demonstrated that0.21l CEL I enzyme cleavage product peak correspondingthereto after diluting the concentration of the PCR product of approximately10ng/mL-27ng/μl.(5) Identified NtPAL4genotypes segregation by CE-TILLING screening systemand found thatMZ2-120line carry homologous mutation whereas MZ2-776lines carry heterozygous mutation withsegregation ratio of1:2:1. MZ2-625lines were detected in the M3generation for the wild-type. |
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