棉铃虫江浦和安阳种群对Cry1Ac和Cry2Ab抗性基因频率及抗性遗传方式
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摘要
表达苏云金杆菌杀虫晶体蛋白的转基因Bt作物在全球范围推广种植,有效控制了重要作物上靶标害虫的为害,同时减少了化学杀虫剂的使用。Bt棉花自1997年开始在我国种植,目前Bt棉花种植面积已经占我国棉花总种植面积70%左右,Bt棉花的大范围推广,有效地抑制了棉花及其他寄主作物上棉铃虫的种群密度。鉴于在其他国家已经有田间害虫对Bt作物产生抗性进化的先例及在我国北方棉区部分田间种群对CrylAc敏感性下降的报道,棉铃虫对Bt棉花的抗性进化一直是Bt棉花持续应用的最大威胁。
目前,主要依据对害虫室内Bt抗性品系的研究结果来制定相应的抗性检测方法和治理措施,高剂量/庇护所和基因叠加是现行的两种主要抗性治理策略。靶标害虫自然种群Bt抗性的进化过程比室内筛选品系复杂,因此,基于对田间种群抗性基因的检测鉴定及其抗性遗传方式的研究对建立更加合理的抗性监测和治理策略具有重要意义。为此,通过F1检测技术对来自长江流域和黄河流域的两个棉铃虫田间种群进行了抗性基因检测,鉴定到大量与室内抗性品系不同的抗性基因,发现了田间种群钙粘蛋白抗性等位基因的多样性。通过建立不同的田间抗性等位基因纯合品系进行抗性遗传互补研究,发现田间种群至少存在2种不同的抗性机制。Cry2Ab是基因叠加转基因棉花的一种重要候选基因,通过对河南安阳棉铃虫田间种群室内筛选获得了Cry2Ab抗性品系,并对其抗性风险进行了评估。本文的研究结果对于我国棉铃虫对Bt棉花抗性的监测和治理具有重要指导意义。
1.棉铃虫江苏江浦种群钙粘蛋白基因突变的多样性
通过F1检测对2008年江苏江浦棉铃虫田间种群进行了与CrylAc抗性相关的钙粘蛋白突变基因频率估算及鉴定。在本试验中,成功检测了123个采集自江苏江浦的田间棉铃虫与室内抗性品系SCD-rl(r1纯合子)杂交单对系的F1后代。在六个候选单对系中,共鉴定到5种不同的钙粘蛋白(Ha_BtR)突变等位基因(r4-r8)。在5个抗性基因中,由于在基因组DNA中发生碱基替换或插入突变而导致基因发生突变。在另外两个候选单对系中,检测到完整的钙粘蛋白等位基因,推测在两个单对系的田间亲本个体上,可能携带由于氨基酸点突变而导致钙粘蛋白受体功能丧失的抗性基因,或者携带其它位点的抗性基因。通过计算,2008年江苏江浦种群钙粘蛋白突变基因频率为0.024(0.010-0.055)。结合之前在棉铃虫中已经发现3个钙粘蛋白突变等位基因(r1、r2和r3)与Cry1Ac抗性相关,目前已经在我国3个棉铃虫田间种群中发现8个不同的钙粘蛋白突变基因,田间突变基因的多样性将严重影响DNA分子检测技术的有效性,F1检测技术用于棉铃虫田间种群的抗性基因检测将更加切实有效。
2.棉铃虫河南安阳种群抗性基因频率检测及钙粘蛋白基因突变鉴定
通过Fl检测对2009年河南安阳棉铃虫田间种群进行了与Cry1Ac抗性相关基因的频率估算及鉴定。在成功检测到的215个单对系中,发现34个候选单对的田间个体可能携带抗性基因,该地区田间棉铃虫种群对Cry1Ac的抗性基因频率为0.091(0.066-0.123)。在候选的阳性单对中,鉴定到胞内区缺失突变与可变剪接两种新型的钙粘蛋白突变基因,进一步丰富了田间钙粘蛋白基因突变的多样性。同时检测到大量正常翻译的钙粘蛋白等位基因,这些基因可能存在氨基酸替换引起的抗性突变。
通过检测阳性单对系F1存活幼虫与室内敏感品系杂交后代,进行田间抗性基因显隐性鉴定,发现86%的田间个体携带隐性抗性基因,其余14%的个体携带非隐性抗性基因。尽管在田间种群中检测到大量个体携带隐性抗性基因,但通过间接和直接法预测田间存活个体的基因型,发现至少携带一个非隐性抗性基因的个体分别占80%和49%。在田间种群的抗性进化中,非隐性抗性基因在决定田间抗性个体频率中的贡献高于隐性抗性基因,因此研究和开发针对非隐性抗性基因的监测技术和抗性治理策略将更加重要和迫切。
3.F1筛选获取的棉铃虫CrylAc抗性等位基因的遗传方式及遗传互补
建立了5个不同的田间抗性等位基因纯合品系Jp14, An1, An29, An357, An156并进行了抗性分析。生物测定显示,与敏感品系SCD相比,5个抗性品系对Cry1Ac活化毒素分别具有128,215,41,881,121倍的抗性,对Cry2Ab毒素无交互抗性。通过与敏感品系的正反交和回交分析表明,Jp14为不完全隐性抗性,An1, An29, An357, An156为不完全显性抗性,5个品系均为常染色体,单基因遗传。通过与室内抗性生品系SCD-rl杂交进行遗传互补分析发现,Jp14, Anl, An29, An357品系中的抗性基因和SCD-r1品系的抗性基因均存在遗传互补作用,进一步验证了F1检测技术的有效性。An156品系可能为其它位点导致的不完全显性抗性。本研究表明：(1)F1筛选可以同时检测到隐性抗性等位基因和非隐性抗性等位基因；(2)棉铃虫钙粘蛋白抗性基因突变的多样性可以导致抗性水平和抗性遗传方式的多样性；(3)在选用抗性监测方法和设计抗性治理策略时,应充分考虑到棉铃虫田间种群中存在的非隐性抗性等位基因。
4.棉铃虫Cry2Ab抗性品系的交互抗性及抗性遗传方式
通过对2009年采自河南安阳的棉铃虫田间种群在室内用Cry2Ab4毒素进行连续筛选,获得了对Cry2Ab4具有高水平抗性的An2Ab品系。An2Ab抗性品系在筛选至第11代时与起始种群An相比,对Cry2Ab4具有262倍的抗性,同时对Cry1Ac活化毒素具有37倍的交互抗性。抗性遗传研究发现,在第11代时,An2Ab品系对Cry2Ab和Cry1Ac毒素同为不完全显性、常染色体遗传,对两种毒素的抗性都为多基因控制。继续用Cry2Ab4筛选的An2Ab品系在第20代时对Cry2Ab抗性升至636倍,同时对Cry1Ac活化毒素具有60倍的交互抗性。这是首次在棉铃虫中发现Cry2Ab抗性品系具有对Cry1Ac的交互抗性。尽管该品系是否可以在表达Cry1Ac和Cry2Ab的双价Bt棉花上存活需要进一步研究,我国棉铃虫具有同时对Cry1Ac和Cry2Ab产生抗性的潜力提示我们在选择应用表达Cry2Ab毒素转基因Bt棉花时应持谨慎态度。
Transgenic crops producing insecticidal protein toxins from Bacillus thuringiensis (Bt) have been adopted worldwide. Planting of Bt crops have provided effective control of target pests, and also reduced the use of chemical insecticides. Bt cotton expressing Cry1Ac protein has been adopted in China since1997, now it accounts for nearly70%of all cotton. The large-scale planting of Bt cotton has been very successful so far not only in controlling Helicoverpa armigera on Bt cotton designed to resist this pest but also in reducing its population density on other host crops. Some pest populations have evolved resistance to Bt crops in the field, and decreased susceptibilty to Cry1Ac in field populations of H. armigera from northern China was associated with intensive Bt cotton planting. The evolution of resistance in field populations of H. armigera to Bt cotton is a major threat to the continued efficacy of Bt cotton in China.
"High dose/refuge" and "pyramiding" are two major resistance management strategies designed for delaying insect resistance development to Bt crops. These two strategies are mainly based on knowledge of Bt resistance from the laboratory-selected strains with resistance to Bt. However, resistance evolution to Bt crops is much more complex in field populations than in laboratory-selected strains. It will be rational to develop resistance detection tactics and resistance management strategies according to resistance nature of field-derived resistant populations. In order to gain a timely understanding of the evolution of resistance of H. armigera to Cry1Ac after continuous cultivation of Bt cotton, we detected resistance alleles to Cry1Ac in two field populations of H. armigera collected respectively from Yellow River Region and Yangtze River Region by using a single pair mating technique (or F1screen). Diverse cadherin alleles conferring Cry1Ac resistance were identified from these two field populations. Two different resistance alleles were detected from five Cry1Ac-resistant strains isolated from field populations. Cry2Ab is an important candidate Bt toxin gene pyramided with CrylAc in the second generation Bt cotton. A Cry2Ab-resistant strain of H. armigera was selected in the laboratory, and cross resistance pattern and inheritance mode of this resistant strain was evaluated. The results from the present study will have a great significance in designing resistance monitoring and management tactics for Bt cotton in China.
1. Diverse cadherin mutations conferring resistance to CrylAc in a field population of H. armigera from Jiangpu of Jiangsu Province
Previous results revealed3null alleles (r1-r3) of a cadherin gene (Ha_BtR) conferring CrylAc resistance in H. armigera. An F1screen of123single pair families was conducted between a Cry1Ac-resistant strain (the SCD-rl strain, homozygous for the r1allele of Ha_BtR) and field-derived insects from Jiangpu population (Jiangsu province, China) in2008. Five new null alleles of Ha_BtR (r4-r8) were identified in six candidate single-pair families. These null alleles were created through either an insertion or a point mutation. Interestingly, intact alleles of Ha_BtR were found in two field-derived insects from another two candidate single-pair families. It suggests that these two field-derived insects may carry novel resistance alleles of Ha_BtR, with missense mutations resulting in a non-functional cadherin protein, or a major dominant mutation at a locus other than cadherin. The resistance allele frequency of Ha_BtR was detected at an appreciable level (0.024) in the Jiangpu population of H. armigera in2008. Together with previous findings, a total of eight different resistance alleles of Ha_BtR were identified from three Chinese strains ofH. armigera. Mutational diversity of Ha_BtR could impair DNA screening for Bt resistance allele frequency in the field, and an F1screen should be used routinely for monitoring cadherin-based resistance allele frequencies in H. armigera.
2. Detection of resistance allele frequency and identification of cadherin mutations in a field population of H. armigera from Anyang of Henan Province
Using an F1screen method, we detected the frequency of alleles conferring resistance to the CrylAc in Anyang population of H. armigera in2009. The field parents of34single-pair families may carry resistance alleles in the215single-pair families tested, and the resistance allele frequency was estimated to be0.091. Two new types of mutational modes of cadherin were detected, namely deletion at the cytoplasmic domain and mis-splicing of cadherin. However, intact alleles of cadherin were present in field-derived insects from most of the candidate single-pair families. It suggests that cadherin can confer resistance to Cry1Ac through amino acid substitutions in cadherin of H. armigera.
Among the29lines generated by crossing survivors from the F1screen with the susceptible SCD strain, bioassay results at the diagnostic concentration of CrylAc showed that for Anyang population,86%of the resistance alleles were recessive cadherin alleles and14%were non-recessive alleles at any locus. Using two methods to estimate genotype frequencies,49to80%of resistant individuals had at least one non-recessive resistance allele. The results here suggest that non-recessive alleles make much more contribution to the resistant individual frequency in Anyang population than the recessive cadherin alleles. Adaptive resistance management strategies are urgently needed to cope with non-recessive resistance alleles in field-selected populations of H. armigera.
3. Inheritance modes and complementation tests with cadherin locus of CrylAc-resistant alleles of H. armigera derived from Fi screens
Five Cryl Ac-resistant strains of H. armigera were established from part of positive single-families of F1screens. The resistance strains Jp14, Anl, An29, An357and An156had128-,215-,41-,881-and121-fold resistance to CrylAc compared respectively to the susceptible SCD strain. All the five strains showed no cross-resistance to Cry2Ab. Through reciprocal crosses between resistant strains and SCD, we found that CrylAc resistance in all five strains was autosomal and controlled by a single locus. CrylAc resistance in Anl, An29, An357and An156was non-recessive, but CrylAc resistance in Jp14was recessive. Genetic complementation tests showed that CrylAc resistance in Jpl4, Anl, An29and An357was cadherin-based, and CrylAc resistance in An156was not cadherin-based. From these results, we concluded:(1) F1screen can detect both recessive resistance alleles and non-recessive resistance alleles in H. armigera;(2) Diverse mutations of cadherin may confer different levels of magnitude and dominance of CrylAc resistance;(3) More efforts are recommended to detect, characterize and design strategies to counter non-recessive resistance alleles of H. armigrea to Bt cotton.
4. Cross resistance and genetics of Cry2Ab resistance in a laboratory-selected strain of H. armigera
Laboratory selection with Cry2Ab against a field population from Anyang produced high levels of resistance to Cry2Ab in An2Ab strain of H. armigera. When An2Ab was selected at the11th generation (G11), it developed262-fold resistance to Cry2Ab and37-fold cross-resistance to Cry1Ac compared to the susceptible progenitor strain (An). Reciprocal crosses between the An and An2Ab at G11showed that resistance to Cry1Ac and Cry2Ab in the An2Ab strain was partially dominant. Backcrossing between the F1progeny and An showed that resistance to Cry2Ab and Cry1Ac in the An2Ab strain was controlled by more than one genes. Resistance to Cry2Ab increased to636-fold at the20th generation (G20) of selection, and cross resistance to Cry1Ac also elevated to60-fold compared to An. This is the first report that Cry2Ab-resistant strain of H. armigera has cross-resistance to Cry1Ac. Although we have not determined if An2Ab strain can survive on the Bt cotton expressing both CrylAc and Cry2Ab toxins, cross resistance potential between Cry2Ab and CrylAc reminds us we should use dual-Bt cotton (Cry2Ab+CrylAc) cautiously as a resistance management tactic in China.
目前,主要依据对害虫室内Bt抗性品系的研究结果来制定相应的抗性检测方法和治理措施,高剂量/庇护所和基因叠加是现行的两种主要抗性治理策略。靶标害虫自然种群Bt抗性的进化过程比室内筛选品系复杂,因此,基于对田间种群抗性基因的检测鉴定及其抗性遗传方式的研究对建立更加合理的抗性监测和治理策略具有重要意义。为此,通过F1检测技术对来自长江流域和黄河流域的两个棉铃虫田间种群进行了抗性基因检测,鉴定到大量与室内抗性品系不同的抗性基因,发现了田间种群钙粘蛋白抗性等位基因的多样性。通过建立不同的田间抗性等位基因纯合品系进行抗性遗传互补研究,发现田间种群至少存在2种不同的抗性机制。Cry2Ab是基因叠加转基因棉花的一种重要候选基因,通过对河南安阳棉铃虫田间种群室内筛选获得了Cry2Ab抗性品系,并对其抗性风险进行了评估。本文的研究结果对于我国棉铃虫对Bt棉花抗性的监测和治理具有重要指导意义。
1.棉铃虫江苏江浦种群钙粘蛋白基因突变的多样性
通过F1检测对2008年江苏江浦棉铃虫田间种群进行了与CrylAc抗性相关的钙粘蛋白突变基因频率估算及鉴定。在本试验中,成功检测了123个采集自江苏江浦的田间棉铃虫与室内抗性品系SCD-rl(r1纯合子)杂交单对系的F1后代。在六个候选单对系中,共鉴定到5种不同的钙粘蛋白(Ha_BtR)突变等位基因(r4-r8)。在5个抗性基因中,由于在基因组DNA中发生碱基替换或插入突变而导致基因发生突变。在另外两个候选单对系中,检测到完整的钙粘蛋白等位基因,推测在两个单对系的田间亲本个体上,可能携带由于氨基酸点突变而导致钙粘蛋白受体功能丧失的抗性基因,或者携带其它位点的抗性基因。通过计算,2008年江苏江浦种群钙粘蛋白突变基因频率为0.024(0.010-0.055)。结合之前在棉铃虫中已经发现3个钙粘蛋白突变等位基因(r1、r2和r3)与Cry1Ac抗性相关,目前已经在我国3个棉铃虫田间种群中发现8个不同的钙粘蛋白突变基因,田间突变基因的多样性将严重影响DNA分子检测技术的有效性,F1检测技术用于棉铃虫田间种群的抗性基因检测将更加切实有效。
2.棉铃虫河南安阳种群抗性基因频率检测及钙粘蛋白基因突变鉴定
通过Fl检测对2009年河南安阳棉铃虫田间种群进行了与Cry1Ac抗性相关基因的频率估算及鉴定。在成功检测到的215个单对系中,发现34个候选单对的田间个体可能携带抗性基因,该地区田间棉铃虫种群对Cry1Ac的抗性基因频率为0.091(0.066-0.123)。在候选的阳性单对中,鉴定到胞内区缺失突变与可变剪接两种新型的钙粘蛋白突变基因,进一步丰富了田间钙粘蛋白基因突变的多样性。同时检测到大量正常翻译的钙粘蛋白等位基因,这些基因可能存在氨基酸替换引起的抗性突变。
通过检测阳性单对系F1存活幼虫与室内敏感品系杂交后代,进行田间抗性基因显隐性鉴定,发现86%的田间个体携带隐性抗性基因,其余14%的个体携带非隐性抗性基因。尽管在田间种群中检测到大量个体携带隐性抗性基因,但通过间接和直接法预测田间存活个体的基因型,发现至少携带一个非隐性抗性基因的个体分别占80%和49%。在田间种群的抗性进化中,非隐性抗性基因在决定田间抗性个体频率中的贡献高于隐性抗性基因,因此研究和开发针对非隐性抗性基因的监测技术和抗性治理策略将更加重要和迫切。
3.F1筛选获取的棉铃虫CrylAc抗性等位基因的遗传方式及遗传互补
建立了5个不同的田间抗性等位基因纯合品系Jp14, An1, An29, An357, An156并进行了抗性分析。生物测定显示,与敏感品系SCD相比,5个抗性品系对Cry1Ac活化毒素分别具有128,215,41,881,121倍的抗性,对Cry2Ab毒素无交互抗性。通过与敏感品系的正反交和回交分析表明,Jp14为不完全隐性抗性,An1, An29, An357, An156为不完全显性抗性,5个品系均为常染色体,单基因遗传。通过与室内抗性生品系SCD-rl杂交进行遗传互补分析发现,Jp14, Anl, An29, An357品系中的抗性基因和SCD-r1品系的抗性基因均存在遗传互补作用,进一步验证了F1检测技术的有效性。An156品系可能为其它位点导致的不完全显性抗性。本研究表明：(1)F1筛选可以同时检测到隐性抗性等位基因和非隐性抗性等位基因；(2)棉铃虫钙粘蛋白抗性基因突变的多样性可以导致抗性水平和抗性遗传方式的多样性；(3)在选用抗性监测方法和设计抗性治理策略时,应充分考虑到棉铃虫田间种群中存在的非隐性抗性等位基因。
4.棉铃虫Cry2Ab抗性品系的交互抗性及抗性遗传方式
通过对2009年采自河南安阳的棉铃虫田间种群在室内用Cry2Ab4毒素进行连续筛选,获得了对Cry2Ab4具有高水平抗性的An2Ab品系。An2Ab抗性品系在筛选至第11代时与起始种群An相比,对Cry2Ab4具有262倍的抗性,同时对Cry1Ac活化毒素具有37倍的交互抗性。抗性遗传研究发现,在第11代时,An2Ab品系对Cry2Ab和Cry1Ac毒素同为不完全显性、常染色体遗传,对两种毒素的抗性都为多基因控制。继续用Cry2Ab4筛选的An2Ab品系在第20代时对Cry2Ab抗性升至636倍,同时对Cry1Ac活化毒素具有60倍的交互抗性。这是首次在棉铃虫中发现Cry2Ab抗性品系具有对Cry1Ac的交互抗性。尽管该品系是否可以在表达Cry1Ac和Cry2Ab的双价Bt棉花上存活需要进一步研究,我国棉铃虫具有同时对Cry1Ac和Cry2Ab产生抗性的潜力提示我们在选择应用表达Cry2Ab毒素转基因Bt棉花时应持谨慎态度。
Transgenic crops producing insecticidal protein toxins from Bacillus thuringiensis (Bt) have been adopted worldwide. Planting of Bt crops have provided effective control of target pests, and also reduced the use of chemical insecticides. Bt cotton expressing Cry1Ac protein has been adopted in China since1997, now it accounts for nearly70%of all cotton. The large-scale planting of Bt cotton has been very successful so far not only in controlling Helicoverpa armigera on Bt cotton designed to resist this pest but also in reducing its population density on other host crops. Some pest populations have evolved resistance to Bt crops in the field, and decreased susceptibilty to Cry1Ac in field populations of H. armigera from northern China was associated with intensive Bt cotton planting. The evolution of resistance in field populations of H. armigera to Bt cotton is a major threat to the continued efficacy of Bt cotton in China.
"High dose/refuge" and "pyramiding" are two major resistance management strategies designed for delaying insect resistance development to Bt crops. These two strategies are mainly based on knowledge of Bt resistance from the laboratory-selected strains with resistance to Bt. However, resistance evolution to Bt crops is much more complex in field populations than in laboratory-selected strains. It will be rational to develop resistance detection tactics and resistance management strategies according to resistance nature of field-derived resistant populations. In order to gain a timely understanding of the evolution of resistance of H. armigera to Cry1Ac after continuous cultivation of Bt cotton, we detected resistance alleles to Cry1Ac in two field populations of H. armigera collected respectively from Yellow River Region and Yangtze River Region by using a single pair mating technique (or F1screen). Diverse cadherin alleles conferring Cry1Ac resistance were identified from these two field populations. Two different resistance alleles were detected from five Cry1Ac-resistant strains isolated from field populations. Cry2Ab is an important candidate Bt toxin gene pyramided with CrylAc in the second generation Bt cotton. A Cry2Ab-resistant strain of H. armigera was selected in the laboratory, and cross resistance pattern and inheritance mode of this resistant strain was evaluated. The results from the present study will have a great significance in designing resistance monitoring and management tactics for Bt cotton in China.
1. Diverse cadherin mutations conferring resistance to CrylAc in a field population of H. armigera from Jiangpu of Jiangsu Province
Previous results revealed3null alleles (r1-r3) of a cadherin gene (Ha_BtR) conferring CrylAc resistance in H. armigera. An F1screen of123single pair families was conducted between a Cry1Ac-resistant strain (the SCD-rl strain, homozygous for the r1allele of Ha_BtR) and field-derived insects from Jiangpu population (Jiangsu province, China) in2008. Five new null alleles of Ha_BtR (r4-r8) were identified in six candidate single-pair families. These null alleles were created through either an insertion or a point mutation. Interestingly, intact alleles of Ha_BtR were found in two field-derived insects from another two candidate single-pair families. It suggests that these two field-derived insects may carry novel resistance alleles of Ha_BtR, with missense mutations resulting in a non-functional cadherin protein, or a major dominant mutation at a locus other than cadherin. The resistance allele frequency of Ha_BtR was detected at an appreciable level (0.024) in the Jiangpu population of H. armigera in2008. Together with previous findings, a total of eight different resistance alleles of Ha_BtR were identified from three Chinese strains ofH. armigera. Mutational diversity of Ha_BtR could impair DNA screening for Bt resistance allele frequency in the field, and an F1screen should be used routinely for monitoring cadherin-based resistance allele frequencies in H. armigera.
2. Detection of resistance allele frequency and identification of cadherin mutations in a field population of H. armigera from Anyang of Henan Province
Using an F1screen method, we detected the frequency of alleles conferring resistance to the CrylAc in Anyang population of H. armigera in2009. The field parents of34single-pair families may carry resistance alleles in the215single-pair families tested, and the resistance allele frequency was estimated to be0.091. Two new types of mutational modes of cadherin were detected, namely deletion at the cytoplasmic domain and mis-splicing of cadherin. However, intact alleles of cadherin were present in field-derived insects from most of the candidate single-pair families. It suggests that cadherin can confer resistance to Cry1Ac through amino acid substitutions in cadherin of H. armigera.
Among the29lines generated by crossing survivors from the F1screen with the susceptible SCD strain, bioassay results at the diagnostic concentration of CrylAc showed that for Anyang population,86%of the resistance alleles were recessive cadherin alleles and14%were non-recessive alleles at any locus. Using two methods to estimate genotype frequencies,49to80%of resistant individuals had at least one non-recessive resistance allele. The results here suggest that non-recessive alleles make much more contribution to the resistant individual frequency in Anyang population than the recessive cadherin alleles. Adaptive resistance management strategies are urgently needed to cope with non-recessive resistance alleles in field-selected populations of H. armigera.
3. Inheritance modes and complementation tests with cadherin locus of CrylAc-resistant alleles of H. armigera derived from Fi screens
Five Cryl Ac-resistant strains of H. armigera were established from part of positive single-families of F1screens. The resistance strains Jp14, Anl, An29, An357and An156had128-,215-,41-,881-and121-fold resistance to CrylAc compared respectively to the susceptible SCD strain. All the five strains showed no cross-resistance to Cry2Ab. Through reciprocal crosses between resistant strains and SCD, we found that CrylAc resistance in all five strains was autosomal and controlled by a single locus. CrylAc resistance in Anl, An29, An357and An156was non-recessive, but CrylAc resistance in Jp14was recessive. Genetic complementation tests showed that CrylAc resistance in Jpl4, Anl, An29and An357was cadherin-based, and CrylAc resistance in An156was not cadherin-based. From these results, we concluded:(1) F1screen can detect both recessive resistance alleles and non-recessive resistance alleles in H. armigera;(2) Diverse mutations of cadherin may confer different levels of magnitude and dominance of CrylAc resistance;(3) More efforts are recommended to detect, characterize and design strategies to counter non-recessive resistance alleles of H. armigrea to Bt cotton.
4. Cross resistance and genetics of Cry2Ab resistance in a laboratory-selected strain of H. armigera
Laboratory selection with Cry2Ab against a field population from Anyang produced high levels of resistance to Cry2Ab in An2Ab strain of H. armigera. When An2Ab was selected at the11th generation (G11), it developed262-fold resistance to Cry2Ab and37-fold cross-resistance to Cry1Ac compared to the susceptible progenitor strain (An). Reciprocal crosses between the An and An2Ab at G11showed that resistance to Cry1Ac and Cry2Ab in the An2Ab strain was partially dominant. Backcrossing between the F1progeny and An showed that resistance to Cry2Ab and Cry1Ac in the An2Ab strain was controlled by more than one genes. Resistance to Cry2Ab increased to636-fold at the20th generation (G20) of selection, and cross resistance to Cry1Ac also elevated to60-fold compared to An. This is the first report that Cry2Ab-resistant strain of H. armigera has cross-resistance to Cry1Ac. Although we have not determined if An2Ab strain can survive on the Bt cotton expressing both CrylAc and Cry2Ab toxins, cross resistance potential between Cry2Ab and CrylAc reminds us we should use dual-Bt cotton (Cry2Ab+CrylAc) cautiously as a resistance management tactic in China.
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