A Linkage Map Of Helicoverpa Armigera (Hübner) Based On AFLP And AFLP Analysis

In chapter 1 of this thesis,we summarized the progress of resistance of transgenic crops to pests,resistance of pests to transgenic Bt crops,strategies of resistance management and AFLP.Chapter 2 involves laboratory selection for transgenic Bt cotton resistant and susceptible strains in cotton bollworm,Helicoverpa armigera(Hübner).Selection experiment of purification and maintenance for high resistance to transgenic Bt cotton expressing CrylAc toxin in population of H.armigera were conducted using a leaf-feeding method.The resistant Level of H.armigera to CrylAc was enhanced from 2523.24-fold to 7310.8-fold by 15 generation of selection.A fenvalerate susceptible strain was developed by selection of progenies that resulted from single-pair matings of cotton bollworm collected from Yanshi Country,Herman Province,under conditions free of insecticides,and its LC₅₀ was 0.14μg/mL.Chapter 3 studied the extraction of complete DNA from H.armigera(Hübner). The analysis of amplified fragment length polymorphism(AFLP) has the potential to become a powerful new DNA fingerprinting technique for studying genetic relationship and genetic diversity in insects.The CTAB based DNA extraction protocol provided DNA suitable for AFLP assay.Its process mainly contained six parts,including digesting of tissue by lysis buffer,precipitation of polysaccharides by CTAB,removal of proteins by chloroform:isoamylalcohol(24:1),precipitation of DNA by PEG,dissolving DNA by TE buffer and consuming of RNA by RNase A. The ratios of A₂₆₀/A₂₈₀ of genomic DNAs gotten by the method were almost between 1.7 and 1.9,and the sizes of products digested by EcoRâ… /Mseâ… were mainly 50-1000bp.Chapter 4 discussed several reagents in polypropylene aroylamino gel electrophoresis(PAGE) and silver staining process,using single reagent to analysis the role played in the process of silver staining.A different result corresponding to the experimental practices were recieved,and AFLP silver staining was effectively solved by reagent quality.Chapter 5 mapped the linkage map of Helicoverpa armigera(Hübner).AFLP makers were mapped using BC1 population.From 50 BC₁[F₁♀(♀_YCR×♂_YCS)×♂_YCR] samples 406 polymorphic markers were detected using a method of AFLP.In this study 23 AFLP primer combinations were used.217 valid loci consistent with the expected segregation ratio of 1:1 at P = 0.05 significance level,might be used in mapping.35 linkage groups with 126 loci were mapped by using Mapmaker/Exp (Version 3.0b),in which we set LOD=3.0 and maximum recombination fraction= 0.3, and Kosambi function.There were 91 unlinked loci in these 217 valid loci and the ratio of mapping loci to valid loci was 58.06%.The number of loci mapped in the 35 groups was at a range of 2～11 and there were 3.6(average value) loci per group.The total length of the groups was 1138.4 cM.The lengths of the35 groups varied at a range of 4.0～166.1 cM.The average length of groups was 32.53 cM.Chapter 6 BtR gene flanked with two AFLP marker,EaaMcta02 and EaaMcta03, on the 15 linkage group in H.armigera,with 10.40 cM and 13.90 cM.