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Impacts Of Natural Refuge On Resistance Evolution To Bt Toxin Cry1Ac And Identification Of Dominant Bt Resistance Genes In Helicoverpa Armigera

Posted on:2018-09-20Degree:DoctorType:Dissertation
Country:ChinaCandidate:L JinFull Text:PDF
GTID:1363330575977171Subject:Agricultural Entomology and Pest Control
Abstract/Summary:PDF Full Text Request
The cotton bollworm Helicoverpa armigera(Hubner)is one of the most serious pests of agriculture that is distributed globally across Europe,Asia,Africa,Australia and America.In China,H.armigera is distributed in any region where the cotton is planted;in addition,the larvae also feed on many other crops such as corn,wheat,peanuts and soybeans and this pest has caused great loss to the agricultural production.Bt cotton expressing Bacillus thuringiensis insecticidal protein Cry 1 Ac has been planted to control field populations of cotton bollworm in China since 1997 and has effectively reduced the damage to cotton and other host crops.In 2015,China planted?3.7 million hectares of Bt cotton at an adoption rate of 96%of total cotton hectarage(3.8 million).The economic gains at the farmer level from Bt cotton were US$17.5 billion during the period 1997 to 2014.However,to be vigilant,H.armigera has the ability to evolve resistance to Cry 1 Ac,and field-evolved resistance to Bt cotton will diminish or even eliminate all the benefits.Although Bt cotton expressing Cry1Ac has been selecting the field populations continuously for a long time,field-evolved resistance to Bt cotton has not been detected in H.armigera.The natural refuges of non-Bt host plants other than cotton such as corn,peanuts,soybeans and others may produce enough susceptible insects and are expected to delay the rapid evolution of resistance.However,rigorous large-scale tests have yet been carried out to validate the effectiveness the natural refuge strategy.The increases in the frequency of resistance to Cry 1 Ac in some field populations from northern China in 2010 provided early warning of resistance.And based on the patterns of field-evolved resistance to Bt crops in some target pests,if no effective resistance management strategy,early warning will become a more serious problem.Therefore,it is urgently needed to accurately assess the effectiveness of natural refuges and seek for strategies to manage Bt resistance of cotton bollworm in China.Apparently,solving these problems requires a thorough understanding of the patterns and the molecular mechanisms of field-evolved resistance in cotton bollworn from China.In this study,the frequency of resistance to Cry 1 Ac in field-collected populations from six provinces in northern China was monitored continuously during 2011 to 2013.The monitoring results coupled with computer simulation revealed the evolutionary patterns of resistance and confirmed the effectiveness of natural refuges for delaying resistance.Considering that nonrecessive resistance increased faster than recessive resistance,two strains of H.armigera with dominant resistance to Cry 1 Ac were isolated from filed populations for further investigation.Both strains had dominant resistance to Cry 1 Ac and minor but significant cross-resistance to Cry2Ab toxin.Compared with recessive resistance traits,dominant resistance may be especially difficult to suppress.And no reported molecular mechanisms can explain the dominant resistance clearly.Therefore,a series of genetic linkage analyses were carried out to map the dominant resistance QTLs.Eventually a single nucleotide mutation in a new candidate gene was identified to be tightly linked to dominant resistance to Cry 1 Ac in H.armigera.The results here will play an important role in monitoring and predicting the frequency of field-resistance,seeking and developing effective proactive resistance management strategies,and improving even reconstructing the mode of action models for Bt toxins.1.Impacts of natural refuge on resistance evolution to CrylAc in H.armigeraThe "High-dose/refuge" strategy is one of the most effective strategies for delaying insect resistance to Bt crops.And the "natural refuge" strategy relying on refuges of non-Bt host plants other than cotton delayed resistance to Bt cotton in cotton bollworm from China.In order to clarify the actual efficacy and the time limit of this strategy and evolutionary patterns of field-resistance,we tested this strategy by comparing predictions from mathematical modeling with data from a large-scale four-year field study of cotton bollworm resistance to Cry1Ac in six provinces of northern China.Bioassay data revealed the percentage of resistant individuals increased from 0.93%in 2010 to 5.5%in 2013.Modeling predicted the percentage of resistant insects would exceed 98%in 2013 without natural refuges,but would increase to only 1.1%if natural refuges were as effective as non-Bt cotton refuges.The observed rate of increase in the frequency of resistance corresponds closely with the rate predicted with a 56%effective refuge.Therefore,the results implied natural refuges delayed resistance,but were not as effective as an equivalent area of non-Bt cotton refuges.The percentage of resistant individuals with nonrecessive resistance alleles increased from 37%in 2010 to 86%in 2014.Also corresponding with predicted with a 56%effective refuge,nonrecessive resistance increased faster than recessive resistance.The average dominance of nonrecessive resistance alleles increased with years.Under the influence of natural refuges,increasing dominance may be the adaptive direction of Bt resistance evolution.The evolution of nonrecessive resistance threatens the continuous efficacy of natural refuges and switching to Bt cotton producing two or more toxins and integrating other tactics could slow further increases in resistance.2.Dominant resistance to Bt cotton and minor cross-resistance to Cry2Ab in H.armigera from ChinaSince the Bt cotton planted in northern China in 1997,the natural refuges have been delaying the evolution of resistance in field populations of cotton bollworm continuously and efficiently.However,the resistance alleles to Cry1Ac showed complex genetic diversity in field populations from northern China and nonrecessive resistance increased faster than recessive resistance.The evolution of nonrecessive resistance threatens the efficacy of natural refuges.In order to characterize nonrecessive resistance,we isolated the resistance alleles with nonrecessive inheritance from two resistant individuals derived from field populations.After continuous selection with high dose of CrylAc,two homozygous resistant strains named QX7 and AY2 were established.Relative to a susceptible strain,the two resistant strains had 460-and 1200-fold resistance to Cry 1 Ac,respectively.Both strains had dominant resistance to a diagnostic concentration of Cry 1 Ac in diet and to Bt cotton leaves containing Cry 1 Ac.Both strains had low,but significant cross-resistance to Cry2Ab(4.2-and 5.9-fold),which is used widely as the second toxin in two-toxin Bt cotton.Compared with recessive resistance,the resistance in the two strains characterized here may be especially difficult to suppress.3.Genetic mapping of dominant resistance to Cry 1 Ac in H.armigeraThe resistant strain AY2 isolated from the field had functionally dominant resistance to Bt cotton.To facilitate identification of dominance resistance genes,we carried out a series of genetic linkage analyses to map the QTLs controlling dominant resistance in AY2.Firstly,we established a backcross family between F1 of AY2×SCD(male)and a susceptible strain SCD(female).Using genome-wide association study,a single QTL on 6.2-11.5Mbp of HaChrlO was identified to be associated to Cry 1 Ac resistance.In order to narrow the span of this QTL and avoid missing other major genes due to lack of abundant samples and SNP markers,we exploited the biphasic nature of lepidopteron genetic linkage to map the resistant genes again.We established reciprocal backcross families between F1 of AY2×GR and a susceptible strain GR.The progeny from backcross families were selected by Cry1Ac to obtain individuals with resistant genes.The results showed two separated QTLs(HaChr09:2.8-4.0Mbp and HaChr10:10.1-11.4Mbp)were identified to control dominant resistance equivalently.These two QTLs were named as HaBtR-Chr09 and HaBtR-Chr10.This is the first research to identify the QTLs responsible for dominant resistance to Bt in insects.Our results will facilitate to identify novel genes of dominant resistance to Cry1Ac and to clarify the mode of action of Cry1Ac in cotton bollworm.4.Identification of a point mutation in the tetraspanin HaTSPANl conferring dominant resistance to CrylAc in H.armigeraThe cotton bollworm can evolve resistance to Cry1Ac with many roads.The mutations in functional receptor genes such as cadherin and ABCC2 transporter can confer about 140-to 1000-fold resistances to Cryl Ac while resistance often tends to be recessive.But little is known about molecular mechanisms of dominant resistance to Bt.In order to construct strains with single resistance gene,we used two specific SNP markers(HaChr09-3.7M and HaChr10-10.69M)to separate the two resistant genes into two different strains:AY2-9(with R gene from Chr09)and AY2-10(with R gene from Chr10).Bioassay data showed the AY2-9 and AY2-10 strains had 200-and 1400-fold resistance to Cryl Ac respectively relative to SCD.We chose progeny from backcross families established between F1 of AY2-10×SCD(male)and SCD(female)to map resistance gene.Through genetic linkage analyses using 7 SNP markers and 363 BC progeny,we identified a single QTL spanned 10.62-10.87Mbp of Chr10.After screening about 20 genes within this 0.25 Mbp QTL,a single amino acid substitution(L31S)in the tetraspanin gene HaTSPANl was identified to be genetically linked to dominant resistance to Cry1Ac in AY2-10.The L31S mutation was predicted within the first transmembrane(TM1)domain of HaTSPAN1.Several strains with dominant resistance to Cry1Ac(LF8,QX7 and SCD423)were detected to carry the L31S mutation in HaTSPAN1.The midgut BBMVs from AY2 larvae can bind Cryl Ac by a similar pattern with SCD.While some inhibitors specific for cell signaling pathways(PI3K and MAPK)can suppress the dominant resistance effectively in AY2.These data imply that HaTSPAN1 may not serve as a functional receptor for Cry1Ac and may be involved in signaling pathways of proliferation and differentiation of stem cells in midgut.This is the first research to identify the gene responsible for dominant Bt resistance by positional cloning.Our results will play an important role in understanding the mode of action of CrylAc,monitoring the frequency of dominant resistance in field populations,and developing effective resistance management strategies in H.armigera.
Keywords/Search Tags:Helicoverpa armigera, CrylAc, dominant resistance, natural refuge, QTL mapping, tetraspanin
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