Detection Of Frequencies Of Resistance Alleles To Cry1AC In Field Populations Of Helicoverpa Armigera And Genetic Mapping Of Cry1AC Resistance In The LF8 Strain

Cotton is one of the important economic crops in China,and its production has ranked first in the world for many years.Therefore,it plays a vital role in the cotton industry.However,the cotton bollworm,Helicoverpa armigera has posed a serious threat to its production.Chemical control had been the main measure to control this pest for many years.However,insecticide sprays caused serious pollution to the environment.More importantly,the biological characteristics of the cotton bollworm make it extremely easy to evolve resistance to all kinds of pesticides.Resistance to pyrethroids led to outbreaks of the species in China in early 1990s across a wide cotton planting area of 400 million hectares.Since 1997,transgenic cotton expressing Bt toxin Cry1 Ac has widely used for controlling cotton bollworm in China.In 2015,the Bt crops grew on more than 3.8 million hectares in China,and it has taken up 96%of the total national cotton planting area.Bt cotton has effectively suppressed some key pests while causing little or no harm to most non-target organisms,reduced insecticide use and increased significant benefits of economy,social and environment.To delay evolution of resistance to Bt cotton in cotton bollworm,the high dose/refuge strategy of resistance management was put forward at the beginning of Bt cotton adoption.Then,the pyramid strategy was employed with planting dual Bt cotton expressing two different Bt Cry toxins.Despite of the implementation of resistance management tactics,the cotton bollworm is evolving resistance to the Bt cotton as a result of long-term selection pressure.This study employed the F₂ screening method to measure the frequencies of Cry1 Ac resistance alleles of cotton bollworm in major cotton areas of China.Then,we characterized the inheritance mode and cross-resistance spectrum of CrylAc resistance in a field-derived LF8 strain of H.armigera.Further,CrylAc resistance in LF8 was mapped between 10.1-11.4 M region on Chromosome 10.Our results will have important implications in guiding Bt resistance management for sustainable use of Bt cotton in China.1.F₂ screen of resistance alleles to CrylAc in field populations of H.armigeraBt cotton expressing CrylAc toxin has been used for 20 years since 1997.To evaluate current status of resistance evolution in the field,an F₂ screen was employed to determine Cryl Ac-resistance allele frequencies in field populations from Xiajin?Shandong province?,Anyang?Henan province?,Langfang?Hebei province?and Shawan?Xinjiang?in 2014 and 2015.In 2014,a total of 63,109,56 and 58 iso-female families in Xiajin,Anyang,Langfang and Shawan were screened respectively,and the resistance allele frequencies was determined to be 0.048,0.034,0.031 and 0 respectively.In 2015,a total of 59,66 and 61 iso-female families in Xiajin,Anyang and Langfang were screened respectively,and the resistance allele frequencies were determined to be 0.102,0.087,0.062 respectively.Resistance allele frequencies of Xiajin,Anyang and Langfang populations in 2015 were doubled when respectively compared to that in 2014,and significant difference between years was observed for Anyang,but not for the other populations.Analyzing data of field populations collected from Anyang between 2009 and 2015,Anyang populations maintain high levels of resistance allele frequencies to CrylAc?0.034-0.087?,and the highest variation between years is 2.6-fold.In summary,CrylAc resistance allele frequencies in populations from northern China are going up steady,but no resistance alleles have been detected in populations from Shawan of Xingjiang.2.Genetic mapping of CrylAc resistance in the LF8 strain of H.armigeraConsidering situations of planting multiple crops together and a large number of small-scale cotton producers,natural refuge strategy has been implemented in China for resistance management of cotton bollworm.Although the natural refuge strategy delayed resistance evolution substantially,resistance allele frequencies are still increasing steadily,especially for dominant resistance alleles.LF8 strain with dominant resistance to CrylAc was isolated from Langfang field population collected in 2014.Compared to a susceptible strain?SCD?,LF8 strain developed 985-fold resistance to CrylAc,serious cross-resistance to Cryl Aa?552-fold?and CrylAb?134-fold?,low but signifiant cross-resistance to Cry2Ab,but no cross-resistance to Vip3Aa.Bioassays of the F1 progeny from reciprocal crosses between LF8 and SCD showed that CrylAc resistance in LF8 was autosomal and dominant.In order to map the resistance loci of LF8 strain,two backcross mapping populations were generated,and a number of molecular makers?SNP?were developed.Our results showed that domiant resistance to CrylAc in LF8 strain was genetically linked with Chromosome 10,and a single locus between10.1-11.4M of Chromosome 10 was tightly linked with CrylAc resistance.No homologous Bt resistance genes were found flanking the resistance locus in Chromosome 10,which suggests there is a novel Bt resistance gene underlying dominant resistance to CrylAc in the LF8 strain of H.armigera.