Detection And Identification Of Cadherin Mutations In A Field Population Of Helicoverpa Armigera From Northern Xinjiang

The cotton bollworm (Helicoverpa armigera Hubner) is one of the most serious pests of global importance. It is a major target pest by transgenic insect-resistant Bt cotton (Bt cotton). Bt cotton has been planted in China since1997. Up to now, the percentage of cotton planted to Bt cotton is more than90%in the Yellow River Valley (YRV) and Changjiang River Valley (CRV) cotton areas, and around10%in Xinjiang cotton area, but has not been planted in northern Xinjiang. Large scale adoption of Bt cotton will accelerate evolution of resistance by the target insect H. armigera. Mutations in H. armigera cadherin gene were proved to be linked with Cryl Ac resistance in the laboratory-selected strains. In field populations of H. armigera from the YRV and CRV cotton areas, diverse mutated cadherin alleles were detected associated with Cryl Ac resistance. In the present study, F1screen was used to detect and identify possible cadherin mutations in a field population (Shawan) from northern Xinjiang, where Bt cotton has not been planted. Our results will help to determine initial frequency and diversity of cadherin mutation, and to make contributions for developing resistance detection methods and resistance management tactics.1. Estabolishment and verification of the F1screen methodThe F1progeny from10single-pair matings in the Cryl Ac-resistant SCD-r1strain (homozygous for r1allele) were screened with1μg/cm2Cry1Ac, survival percentages of these10single-pair families were all more than90%. The F1progeny from10single-pair matings between the Cry1Ac-resistant SCD-r1strain and susceptible SCD strain were screened with1μg/cm2Cry1Ac, survival percentages of these10single-pair families were all less than10%. The dosage1μg/cm2of Cry1Ac was then choosed as the discriminating dose for F1screen. Based on our previous results, survival ranges for different cadherin genotypes were determined as:(1) survival percentage of an F1progeny was<30%, the field-derived male moth carrying the wild allele of cadherin;(2) survival percentage was between30%～70%, the field-derived male moth carrying a single mutated cadherin allele;(3) survival percentage was>70%, the field-derived male moth carrying two mutated cadherin alleles.In order to validate the proposed criterion, single-pair matings between field-collected male moths from Shawan population in2009and SCD-rl strain female moths were prepared, and their F1progeny were treated with1μg/cm2Cry1Ac. The F1progeny from one single pair family (SW144) showed44.4%survival at the discriminating dose of Cry1Ac, suggesting the field-derived male moth may carry a single mutated allele of cadherin. The full-length cadheirn cDNA was cloned and sequenced from the F1survivors, and a mutated allele with a96bp deletion at position781bp was identified, which resulted premature stop at positon196aa. These results confirmed that the F1screen method we set up was both viable and reliable.2. Detection and identification of mutated cadherin alleles of Shawan population from northern XinjiangIn2010,208single-pair matings were prepared between male moths from Shawan population and female moths of SCD-rl strain, and105single-pair matings produced fertile eggs. F1offsprings of each single-pair families were subjected to1μg/cm2Cry1Ac treatment, and6candidates were identified to be heterozygous for cadherin locus. The survivors of these6candidate families were crossed with the susceptible SCD strain, and their F1offsprings were treated1μg/cm2Cry1Ac and no survivors were found. It confirmed the recessive nature of the resistant locus in the field-derived male moths. The full-length cadheirn cDNAs were cloned and sequenced from the F1survivors of6candidate families. A mutated cadhein allele from SW60family was identified with a348bp deletion at positon23bp, producing an116aa deletion. The second mutated cadhein allele from SW80family was identified with a12bp deletion at positon, producing an4aa deletion. The third mutated cadhein allele from SW84family was identified with a G to A mutation at position3275bp, creating a premature stop codon. However, cadhein alleles from SW2, SW33and SW155families were intact, but with amino acid substitutions. It is suggested that amino acid substitutions in the cadherin may confer high levels of resistance to CrylAc in H. armigera. As the results reported from field populations of YRV and CRV cotton areas, Shawan populations from northern Xinjiang also showed diverse cadherin mutations associated with CrylAc resistance.From the F1screen results, resistance allele frequency in Shawan population from northern Xinjiang was estimated to be0.028. Considering Bt cotton has not been adopted in norther Xinjiang, Shawan population had relatively high initial resistance allele frequency. It is indicated that Shawan population of H. armigera may have high risk to evolve resistanc to Bt cotton in future.