Risk Assessment And Molecular Genetic Mechanisms Of Resistance To Cry1ac Plus Cry2ab In Helicoverpa Armigera

Cotton bollworm Helicoverpa armigera（Hübner）is one of the most important pests in the world.This pest has several biological characteristics contributed to its major pest status,including polyphagy,high mobility,high fecundity and strong potential of resistance development.In the 1990s,H.armigera outbreaks were frequently experienced in the main cotton planting areas of China,and caused serious economic losses.Since 1997,China has mainly depended on planting Bt cotton only expressing Cry1Ac to control the damage of H.armigera,which effectively suppressed the field population.The long-term use of the single-gene Bt cotton（Cry1Ac）has led to an increase in the frequency of Cry1Ac resistant individuals in the field populations of H.armigera in Northern China in recent years,and the resistance is evolving to be more dominant,which will compromise and even diminish the effectiveness of the natural refuge strategy we are employed."Gene pyramid"is another main strategy for Bt resistance management,that is,two or more Bt genes with different modes of action for the same target pest are pyramided into the same plant to produce two-gene Bt varieties and three-gene Bt varieties to achieve"redundant killing",thus delay the development of resistance.In the United States,Australia and India,two-gene Bt cotton（Cry1Ac+Cry2Ab）and even three-gene Bt cotton（Cry1Ac+Cry2Ab+Vip3Aa）have been widely planted for resistance management.However,some target pests have evolved resistance to the pyramided Bt cotton in India.Two-gene Bt cotton（Cry1Ac+Cry2Ab）has also been developed in China,so it is necessary to assess the resistance risk of H.armigera to the Bt pyramids,and to investigate the molecular mechanisms of H.armigera resistant to two different Bt proteins,so as to provide an important basis for long-term and sustainable planting of the two-gene Bt cotton in China.In this thesis,the possible resistance risk of H.armigera to Cry1Ac plus Cry2Ab was assessed from three aspects:to ascertain whether H.armigera strains that had high levels of resistance to Cry1Ac are cross-resistant to Cry2Ab;to determine the sensitivity of H.armigera field populations to a discriminating dose of Cry1Ac+Cry2Ab;to evaluate the realized heritability of resistance through selection of the dual Bt resistant strain（SCD-DR）of H.armigera with Cry1Ac+Cry2Ab.Then,the resistance genes of SCD-DR strain were genetically mapped.Two QTLs（located in Chr6 and Chr15）were linked to Cry1Ac resistance,one QTL（Chr23）was linked to Cry2Ab resistance,and one QTL（located in Chr9）was linked to both Cry1Ac and Cry2Ab resistance.Finally,the identification and functional verification of three of the mapped QTLs showed that the Cry1Ac resistant QTL on Chr6 is related to the r1 mutaion of the cadherin gene（Ha Cad）,and the Cry1Ac resistant QTL on Chr15 is irrelevant to Ha ABCC2 and Ha ABCC3,and the QTL of cross resistance to Cry1Ac and Cry2Ab located on Chr9 is associated with a serine protease gene cluster.The specific results are as follows:1.Risk assessment of resistance to Cry1Ac plus Cry2Ab in H.armigeraFirstly,susceptibility levels to Cry2Ab were evaluated in four Cry1Ac resistant strains of H.armigera（with different resistance mechanisms）.Compared with susceptible strain SCD,the resistance ratios of SCD-r1（Ha Cad mutation）,SCD-KI（Ha TSPAN1 mutation）and LF256（unknown mechanism）to Cry1Ac were between 145-and 540-fold,but the susceptibility levels to Cry2Ab were not significantly different（1.2-to 1.4-fold）.The C2/C3-KO strain（Ha ABCC2 and Ha ABCC2 knockout）is more than 5160-fold resistant to Cry1Ac,but 5-fold negative cross-resistance to Cry2Ab.Secondly,7 fields populations of H.armigera collected from Northern China in 2016 were bioassayed with the diagnostic dose of mixed Bt toxin（0.02μg/cm2 Cry1Ac+0.42μg/cm2 Cry2Ab）.The results showed that the resistant individual frequencies of field population to Bt mixed toxin was very low（0.0002～0.01）.Finally,the susceptible strain SCD was screened with the mixed Bt toxin to assess resistance risk.After 15 generations of selection,resistance levels of the selected SCD-DR strain to Cry1Ac and Cry2Ab were 1334-and 118-fold,respectively.Resistance realized heritability of the SCD-DR strain to Cry1Ac and Cry2Ab were 0.25 and 0.11,which were calculated by threshold trait analysis.This result indicated that H.armigera was more readily to evolve resistance to Cry1Ac than Cry2Ab under the joint selection of these two toxins.Meanwhile,the survival of SCD-DR larvae on Bt cotton was significantly higher than that of the SCD strain.Genetic analysis showed that the resistance of SCD-DR strain to these two toxins is autosomal and incompletely dominant,and there is a correlation between Cry1Ac and Cry2Ab resistance（P=0.048<0.05）.These results indicated that the resistance of SCD-DR to Cry1Ac and Cry2Ab is conferred by multiple factors,in addition to the independent resistance factors to Cry1Ac or Cry2Ab,there is also a common factor contributing resistance to both Cry1Ac and Cry2Ab.The above results show that although the Bt cotton expressing Cry1Ac has been planted for a long time in China,the evolved resistance of H.armigera to Cry1Ac does not affect the toxicity of Cry2Ab.Thus,the Bt cotton expressing Cry1Ac and Cry2Ab can be used to manage Bt resistance of H.armigera in China.However,the results from laboratory Bt screening show that H.armigera has the potential to evolve high level of resistance to both Cry1Ac and Cry2Ab.Therefore,when planting Bt cotton,it is necessary to set up refuges and plant non-Bt insect-resistant cotton varieties in order to realize sustainable use of Bt cotton.2.Genetic mapping of resistance to Cry1Ac and Cry2Ab in the SCD-DR strain of H.armigeraFor unknown resistance mechanisms,genetic linkage analyses to map the QTLs are widely used to locate Bt resistance genes and evaluate their contributions for lepidopteran pests.The traditional biphasic mapping method takes advantage of the biphasic nature of lepidopteran genetic linkage to construct two populations for mapping Bt resistance genes.With the development of next generation sequencing and analysis techniques,BSA-seq provides a new way for mapping of Bt resistance genes more efficiently.In this study,Bt resistance QTLs were identified through BSA-seq mapping for the SCD-DR strain.The QTLs on 0～4 Mbp of Ha Chr06,4～8 Mbp of Ha Chr09,and 6～10 Mbp of Ha Chr15 were identified to be associated with Cry1Ac resistance,and the QTLs on 7.5～9.5 Mbp of Ha Chr09 and 7～9 Mbp of Ha Chr23 were identified to be associated with Cry2Ab resistance.Considering that the 7.58 Mbp on Ha Chr09 is linked to both Cry1Ac and Cry2Ab resistance after further fine-mapping,it is suggested that there may be a cross-resistance gene in this overlapped region on Ha Chr09.The above mapping results are helpful to identify candidate genes for further functional verifications.3.Identification and functional verification of Cry1Ac and Cry2Ab resistance genes in the SCD-DR strain of H.armigeraPrevious studies have shown that mutations in Ha Cad（recessive,located on Ha Chr06）,both Ha ABCC2 and Ha ABCC3（recessive,located on Ha Chr15）and Ha TSPAN1（dominant,Ha Chr10）of H.armigera can confer resistance to Cry1Ac,while Ha ABCA2 mutations（recessive,Ha Chr17）cause Cry2Ab resistance in H.armigera.In view of the fact that Cry1Ac resistance of the SCD-DR strain is linked to Ha Chr06 and Ha Chr15,Ha Cad,Ha ABCC2 and Ha ABCC3 should be examined firstly.Through gene cloning,sequence alignment and quantitative analysis of gene expression,the r1 allele of Ha Cad was identified on Ha Chr06,while the resistance QTL on Ha Chr15 is irrelevant to Ha ABCC2and Ha ABCC3.At the same time,Ha ABCA2 on Ha Chr17 is not related to Cry2Ab resistance.Genetic mapping showed that the 7.58 Mbp region on Ha Chr09 of SCD-DR was linked to both Cry1Ac and Cry2Ab resistance,so there is a cross-resistance QTL in this area.Based on the analysis of the genes in this region,there is a serine protease gene cluster composed of 8 genes was found here,which are highly expressed in the midgut of H.armigera larvae.After cloning and sequence alignment of 8 genes in this gene cluster,it was found that the E20G mutation of the serine protease homologue gene Ha OG200431may be related to the resistance of two Bt toxins.Using CRISPR/Cas9 to knock out Ha OG200431 in SCD-DR,and the homozygous knockout strain were crossed with SCD and SCD-DR respectively to establish four strains(6^RR9^SS,6^RR9^RR,6^SS9^RR,6^SS9^SS)with different genotypic combinations.The bioassay results of the above four strains showed that Ha OG200431 of SCD-DR contributes about 4.3-fold and 9.6-fold resistance to Cry1Ac and Cry2Ab,respectively.The Ha OG200431 wild type and mutant proteins expressed in vitro could neither bind to these two Bt toxins nor degrade the toxins.The mechanism of cross-resistance to Cry1Ac and Cry2Ab caused by the mutation of Ha OG200431 needs to be further studied in future.In this thesis,resistance risk to Cry1Ac plus Cry2Ab in H.armigera was systematically assessed.The assessment results provide a necessarydemonstration for the feasibility and prospect of planting two-gene Bt cotton in China.At the same time,through mapping and identification of the resistance genes of the resistant strain with dual Bt resistance,a gene cluster linked to Cry1Ac and Cry2Ab cross resistance was found for the first time,which provides a new perspective for the study of Bt cross resistance mechanism.