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Entomology, Department of
2011
A single major QTL controls expression of larval Cry1F resistance A single major QTL controls expression of larval Cry1F resistance
trait in trait in
Ostrinia nubilalisOstrinia nubilalis
(Lepidoptera: Crambidae) and is (Lepidoptera: Crambidae) and is
independent of midgut receptor genes independent of midgut receptor genes
Brad S. Coates
USDA-ARS
, brad.coates@ars.usda.gov
Douglas V. Sumerford
Iowa State University
Miriam D. Lopez
Iowa State University
Haichuan Wang
University of Nebraska-Lincoln
, hwang4@unl.edu
Lisa M. Fraser
Iowa State University
See next page for additional authors
Follow this and additional works at: https://digitalcommons.unl.edu/entomologyfacpub
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Coates, Brad S.; Sumerford, Douglas V.; Lopez, Miriam D.; Wang, Haichuan; Fraser, Lisa M.; Kroemer,
Jeremy A.; Spencer, Terrence; Kim, Kyung S.; Abel, Craig A.; Hellmich, Richard L.; and Siegfried, Blair D., "A
single major QTL controls expression of larval Cry1F resistance trait in
Ostrinia nubilalis
(Lepidoptera:
Crambidae) and is independent of midgut receptor genes" (2011).
Faculty Publications: Department of
Entomology
. 345.
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Authors Authors
Brad S. Coates, Douglas V. Sumerford, Miriam D. Lopez, Haichuan Wang, Lisa M. Fraser, Jeremy A.
Kroemer, Terrence Spencer, Kyung S. Kim, Craig A. Abel, Richard L. Hellmich, and Blair D. Siegfried
This article is available at DigitalCommons@University of Nebraska - Lincoln: https://digitalcommons.unl.edu/
entomologyfacpub/345
A single major QTL controls expression of larval Cry1F resistance
trait in Ostrinia nubilalis (Lepidoptera: Crambidae)
and is independent of midgut receptor genes
Brad S. Coates
•
Douglas V. Sumerford
•
Miriam D. Lopez
•
Haichuan Wang
•
Lisa M. Fraser
•
Jeremy A. Kroemer
•
Terrence Spencer
•
Kyung S. Kim
•
Craig A. Abel
•
Richard L. Hellmich
•
Blair D. Siegfried
Received: 10 July 2010 / Accepted: 9 June 2011 / Published online: 6 August 2011
Ó Springer 2011
Abstract The European corn borer, Ostrinia nubilalis
(Lepidoptera: Crambidae), is an introduced crop pest in
North America that causes major damage to corn and
reduces yield of food, feed, and biofuel materials. The
Cry1F toxin from Bacillus thuringiensis (Bt) expressed in
transgenic hybrid corn is highly toxic to O. nubilalis larvae
and effective in minimizing feeding damage. A laboratory
colony of O. nubilalis was selected for high levels of Cry1F
resistance ([12,000-fold compared to susceptible larvae)
and is capable of survival on transgenic hybrid corn.
Genetic linkage maps with segregating AFLP markers
show that the Cry1F resistance trait is controlled by a
single quantitative trait locus (QTL) on linkage group 12.
The map position of single nucleotide polymorphism
(SNP) markers indicated that midgut Bt toxin-receptor
genes, alkaline phosphatase, aminopeptidase N, and cad-
herin, are not linked with the Cry1F QTL. Evidence sug-
gests that genes within this genome interval may give rise
to a novel Bt toxin resistance trait for Lepidoptera that
appears independent of known receptor-based mechanisms
of resistance.
Keywords Quantitative trait locus (QTL) Amplified
fragment length polymorphism (AFLP) Single nucleotide
polymorphism (SNP)
Introduction
The development of phenotypic resistance in insect popu-
lations to biopesticides is a concern for crop production
efforts to meet the food, fiber and biofuel needs. Biopes-
ticides have been developed from protein-based crystalline
(cry) toxins endogenously expressed by the soil bacterium
Bacillus thuringiensis (Berliner) (Bt) and shown to be
highly toxic to larval Lepidoptera. The biological activity
of cry toxins is specific, and shows little or no toxicity to
humans, most beneficial insects, and other non-target
organisms (O’Callaghan et al. 2005; Romeis et al. 2006).
Bacillus thuringiensis spores have been used for foliar
applications to crop plants to provide suppression of
feeding damage by susceptible larvae, but the method is
used rarely in the U.S. due to poor environmental stability
of the protein toxin and difficulty timing application with
peak pest populations (Lambert and Peferoen 1992). The
effectiveness of Bt toxins for the control of larval feeding
damage was enhanced through the development of
Electronic supplementary material The online version of this
article (doi:10.1007/s10709-011-9590-0) contains supplementary
material, which is available to authorized users.
B. S. Coates D. V. Sumerford M. D. Lopez
J. A. Kroemer C. A. Abel R. L. Hellmich
USDA-ARS, Corn Insects & Crop Genetics Research Unit,
Genetics Laboratory, Iowa State University, Ames,
IA 50011, USA
B. S. Coates (&)
USDA-ARS, Corn Insects & Crop Genetics Research,
113 Genetics Lab, Iowa State University, Ames, IA 50010, USA
e-mail: brad.coates@ars.udsa.gov
D. V. Sumerford M. D. Lopez L. M. Fraser
C. A. Abel R. L. Hellmich
Department of Entomology, Iowa State University, Ames,
IA 50011, USA
H. Wang T. Spencer B. D. Siegfried
Department of Entomology, University of Nebraska, Lincoln,
NE, USA
K. S. Kim
Department of Agricultural Biotechnology, Seoul National
University, Seoul 151-921, Republic of Korea
123
Genetica (2011) 139:961–972
DOI 10.1007/s10709-011-9590-0
This article is a U.S. government work, and is not subject to copyright in the United States.
genetically-engineered (GE) Bt maize hybrids that express
a high dose of Bt toxins throughout most of the growing
season (Koziel et al. 1993). GE maize hybrids that express
Cry1Ab and Cry1F were respectively released for use by
crop producers in 1996 and 2002, prior to which larvae of
the species Ostrinia nubilalis, the European corn borer,
were arguably the single most destructive pest insect
impacting maize production in the U.S. (Archer et al.
2000). By 2009, GE Bt maize hybrids comprised *46% of
the 35.2 million ha that were planted, with some maize
production areas of the Midwestern U.S. having [50%
(USDA NASS 2009). Since insect control obtained by use
of Bt crops has rapidly become an integral component of
maize production systems, the development of Bt toxin
resistant phenotypes among target insect populations is
predicted to have a high economic cost to crop producers
and consumers.
Currently, GE maize hybrids provide an effective means
to reduce levels of feeding damage by larval O. nubilalis
with a concomitant reduction in use of traditional neurotoxic
chemical insecticides. Although O. nubilalis resistance to Bt
toxins is not observed in field environments, varying levels
of resistance to Cry1Ab (Huang et al. 1997; Bolin et al.
1999; Chaufaux et al. 2001; Siqueira et al. 2006; Coates et al.
2007; Crespo et al. 2009) and Cry1F toxins was observed
following laboratory selection (Pereira et al. 2008a, b).
Additionally, resistance to Bt toxins has been reported in the
lepidopteran species Helicoverpa armigera (Gahan et al.
2001) and Pectinophora gossypiella under laboratory con-
ditions (Morin et al. 2003). Although not an equivalent
representation of field conditions due to reductions in
effective population sizes and exposure to initial low tox-
icity environments (Harshman and Hoffmann 2000), labo-
ratory selections show that target insects have the genetic
potential for developing Bt resistant phenotypes (Ferre
´
and
Van Rie 2002). Realization of this genetic potential was
shown via emergence of populations that are resistant to
field-exposed levels of foliar or transgenic Bt toxins, and
include species Plodia interpunctella (McGaughey 1985),
Plutella xylostela (Tabashnik et al. 1990), Tricoplusia ni
(Janmaat and Myers 2003), Busseola fusca (van Rensburg
2007), and Spodoptera frugiperda (Matten et al. 2008).
Although significant levels of field damage have not been
reported, Tabashnik (2008) suggest resistance to GE cotton
expressing Cry1Ac toxin has emerged in Helicove
rpa zea
populations based on analysis of more than a decade of
phenotypic monitoring data.
Despite the heavy reliance on Bt toxins for pest insect
control, the genetic mechanism(s) of resistance within the
insect pest populations are not well understood, such that
evaluation of current insect resistance management (IRM)
strategies to delay the evolution of resistance phenotypes
cannot be properly evaluated (Glasser and Matten 2003).
Historically, the frequent high dose application of insecti-
cides for insect pest control has led to the selection for
individuals of a population with traits that confer resistance
(Georghiou and Lagunes-Tejeda 1991). The genetic basis
for chemical insecticide tolerance tends to reside in an
increased ability of resistant insects to detoxify the chem-
ical agent, or amino acid changes in receptor proteins that
alter ligand binding affinities. Insecticide chemistries tend
to show a high fidelity for binding specific receptor mol-
ecules that result in insect toxicity and death, but also often
results in reduced toxicity when receptor mutations are
encountered within insect populations (Casida and Quistad
2004). For example, the enzyme acetylcholinesterase is
bound and rendered inactive by insecticides that utilize
organophosphate and carbamate chemistries, but enzyme
insensitivity within an insect population can be achieved by
mutation of a single amino acid (Williamson et al. 1996).
Similarly, a single amino acid alteration in the gamma-
aminobutyric acid (GABA) receptor results in resistance of
Drosophila to cyclodiene insecticides (ffrench-Constant
et al. 1993).
In contrast to the target-specificity typical of chemical
insecticides, there appears to be a heterogeneous set of
protein–protein interactions that occurs within the larval
midgut between an ingested Bt cry toxin and multiple
membrane-bound glycoprotein receptors. The midgut pro-
tein receptors alkaline phosphatase, aminopeptidease N,
and cadherin-like proteins have been implicated as key
factors in Bt toxin modes of action for lepidopteran larvae
(Vadlamudi et al. 1993; Knight et al. 1994; Francis and
Bulla 1997; Jurat-Fuentes et al. 2002). Additionally,
knockout of a b-1,3 galactosyltransferase from the mu-
tagenized Bt resistant 5 (bre5) line of Caenorhabditis
elegans resulted in resistance to Bt toxins (Griffitts et al.
2001), which reiterated the role that postranslational gly-
cosylation may play in toxin-binding of receptor proteins
(Knowles et al. 1991; Jurat-Fuentes et al. 2002). Mutations
in the receptors, aminopeptidase N and cadherin, were also
shown to result in larval Bt toxin-resistance traits among
species of Lepidoptera (Gahan et al. 2001; Morin et al.
2003; Herrero et al. 2005; Xie et al. 2005; Zhang et al.
2009). In contrast, analysis of the Cry1Ac- and Cry2Aa-
resistant CP73 strain of Heliothis virescens indicated that
neither cadherin nor aminopeptidase N receptor genes
contributed to resistance traits (Gahan et al. 2005; Heckel
et al. 2007). Gahan et al. (2005) further indicated that the
H. virescens Cry2Aa resistance trait is determined by more
than one genetic locus. Baxter et al. (2008)
showed that a
single QTL that conferred Plutella xylostella resistance to
Cry1A toxins segregated independently of genome posi-
tions that encode the known glycoprotein receptors of Bt
toxins. Similarly, single nucleotide polymorphism (SNP)
markers for aminopeptidase N or cadherin were shown not
962 Genetica (2011) 139:961–972
123
to co-segregate with Cry1Ab resistance in O. nubilalis
(Coates et al. 2008a). These studies suggest that Bt resis-
tance among Lepidoptera may evolve by multiple inde-
pendent genetic mechanisms (Griffitts and Aroian 2005;
Baxter et al. 2005; Heckel et al. 2007).
Previous investigation of O. nubilalis larval resistance to
the Bt toxin Cry1Ab used SNP markers to track the seg-
regation of the candidate genes aminopeptidase N, braini-
ac, and cadherin among full-sib F
1
s, but was unable to
correlate the inheritance of alleles from resistant parents
with the resistance traits (Coates et al. 2005, 2007, 2008a).
These studies highlighted the limitations of investigating a
small number of genomic loci, and of the need for genome-
wide scans to effectively isolate quantitative trait loci
(QTL). In the current study, we used an O. nubilalis lab-
oratory colony with a [12,000-fold increase in resistance
to the Bt toxin Cry1F (Pereira et al. 2008a), and showed
recessive inheritance controlled from a single genetic locus
(Pereira et al. 2008b). Moreover, the trait conferred larval
survival on transgenic maize hybrids that expressed Cry1F
toxin (Pereira et al. 2008a). In the following, we report the
genetic analysis of O. nubilalis backcross progeny that
survived a high dosage of Cry1F toxin and identify QTL
that contribute to expression of the trait. This analysis was
paired with analysis of segregating markers within genes
that encode candidate Bt toxin-receptors, and include the
midgut membrane-bound glycoproteins alkaline phospha-
tase, aminopeptidase N, and cadherin. Results are signifi-
cant since the single major QTL is segregates independent
of known Bt toxin-receptor genes, and suggests that
O. nubilalis Cry1F resistance has evolved by a novel bio-
chemical mechanism.
Materials and methods
Larval Cry1F toxin-resistance phenotype, pedigrees,
and DNA extraction
A laboratory colony with a [12,000-fold increased Cry1-
toxin tolerance compared to susceptible controls and is
capable of surviving on transgenic corn plants was previ-
ously selected (Pereira et al. 2008a). The trait also was shown
to be controlled by a single genetic locus that is inherited in a
recessive fashion (Pereira et al. 2008b). The sex determina-
tion system in O. nubilalis, and all Lepidoptera, is comprised
of a homogametic sex chromosome pair in males (ZZ) and a
heterogametic pair in females (ZW; Traut and Marec 1997),
where achiasmatic females produce gametes that do not
undergo meiotic recombination (Traut 1977). A biphastic
linkage mapping approach was used as described by Heckel
et al. (1999) to establish pedigrees initiated from a single
cross between a Cry1F resistant female (rr$;P
rr$
) 9 sus-
ceptible male (SS#;P
SS#
), and subsequent backcross fami-
lies were derived from an F
1
male 9 Bt resistant female
(F
1rS#
9 BCP
rr$
; pedigree FQ4) or reciprocal F
1
female 9
resistant male (F
1rS$
9 BCP
rr#
; pedigree FQ5; Fig. 1). Due
to the recessive single locus inheritance of the Cry1F toxin
Fig. 1 Biphasic pedigree and Cry1F toxin bioassay design used to
assess larval resistance traits in O. nubilalis. The family was initiated
from a single parental (P) mate pair between a resistant female
(P
rr$
) 9 susceptible male (P
SS#
), followed by reciprocal backcrosses
between F
1
to resistant line parents (BCP; F
1rS#
9 BCP
rr$
in pedigree
family FQ4; BCP
rr#
9 F
1rS$
in pedigree family FQ5). A total of 44
larvae from each FQ4 and FQ5 were reared on non-Cry1F toxin diet
(normal diet), and remaining backcross progeny subjected to diag-
nostic Cry1F toxin bioassay
Genetica (2011) 139:961–972 963
123
