Genes and genetic mechanisms contributing to fall armyworm
resistance in maize
Marilyn L. Warburton1, Sandra W.
Woolfolk2, J. Spencer Smith2, Leigh
K. Hawkins2, Lina Castano-Duque3,
Matthew D. Lebar3, and W. Paul
Williams2
1USDA ARS Plant Germplasm Introduction and Testing
Research Unit, Pullman, WA
2USDA ARS Corn Host Plant Resistance Research Unit,
Mississippi State, MS
3USDA ARS Food and Feed Safety Research Unit, New
Orleans, LA
Abbreviations: BLUE (Best Linear Unbiased Estimator); CIMMYT
(International Maize and Wheat Improvement Center, in Spanish); CHPRRU
(Corn Host Plant Resistance Research Unit); FAW (fall armyworm); GBS
(genotyping by sequencing); GLM (general linear model); GWAS
(genome-wide association study); MAF (minor allele frequency); MLM
(mixed linear model); PAST (pathway analysis study tool); QTL
(quantitative trait loci).
Abstract
Maize (Zea mays L.) is a crop of major economic and food security
importance globally. The fall armyworm (FAW), Spodoptera
frugiperda, can devastate entire maize crops, especially in countries
or markets that do not allow the use of transgenic crops. Host-plant
insect resistance is an economical and environmentally benign way to
control FAW, and this study sought to identify maize lines, genes, and
pathways that contribute to resistance to FAW. Of 289 maize lines
phenotyped for FAW damage in artificially infested, replicated field
trials over three years, 31 were identified with good levels of
resistance that could donate FAW resistance into elite but susceptible
hybrid parents. The 289 lines were genotyped by sequencing to provide
SNP markers for a genome-wide association study (GWAS), followed by a
metabolic pathway analysis using the Pathway Association Study Tool
(PAST). GWAS identified 15 SNPs linked to 7 genes, and PAST identified
multiple pathways, associated with FAW damage. Top pathways, and thus
useful resistance mechanisms for further study, include hormone
signaling pathways and the biosynthesis of carotenoids (particularly
zeaxanthin), chlorophyll compounds, cuticular wax, known antibiosis
agents, and 1,4-dihydroxy-2-naphthoate. Targeted metabolite analysis
confirmed that maize genotypes with lower levels of FAW damage tend to
have higher levels of chlorophyll a than genotypes with high FAW damage,
which also tend to have lower levels of pheophytin, lutein, chlorophyll
b and β-carotene. The list of resistant genotypes, and the results from
the genetic, pathway, and metabolic study, can all contribute to
efficient creation of FAW resistant cultivars.
Introduction
Maize (Zea mays L.) provides food, feed, and industrial
ingredients to the economy of the United States, worth many billions of
dollars annually. It is a major staple food crop globally as well, and
total grain production was 1,266 million tons in 2019 (World Data Atlas,
Knoema.com). The fall armyworm (FAW), Spodoptera frugiperda (J.
E. Smith) (Lepidoptera: Noctuidae), originally evolved with maize in the
tropical and subtropical regions of the Americas, and although it feeds
on a broad range of host plants, it prefers gramineous plants including
maize, and can devastate a maize crop in a short time (Overton et al.,
2021). It has now spread to almost every country of the Americas,
Africa, and Asia as an invasive and destructive species (Goergen et al.,
2016; FAO 2021). This is of particular concern in Africa, where many
countries do not allow the use of transgenic crops, removing some insect
control options from farmers, but new genetic editing techniques may be
more acceptable to the public (Turnbull et al., 2021).
Native resistance, or host-plant insect resistance conferred by
naturally occurring maize genes, is an economical and environmentally
benign way to control FAW. Maize lines containing native resistance
genes were created in the 1970s through 1990s by the International Maize
and Wheat Improvement Center (CIMMYT) in Mexico (Mihm et al., 1988). The
USDA-ARS Corn Host Plant Resistance Research Unit developed and released
several resistant temperate maize inbred lines; these include Mp496,
Mp701-708, Mp713, Mp714, and Mp716 (Scott and Davis, 1981; Scott et al.,
1982; Williams and Davis, 1980, 1982, 1984, 2000, 2002; Williams et al.,
1990). Although high levels of FAW infestation are better controlled by
transgenic hybrids containing the Bt gene than conventional hybrids with
native resistance, both transgenic and conventionally bred resistant
maize hybrids are significantly less damaged by FAW feeding than
non-resistant commercial hybrids (Williams et al., 1997).
Native resistance to insects in maize is generally quantitative in
nature, governed by many genes, rather than a single gene conferring
most or all of the resistance (McMullen et al., 2009). Quantitative
trait loci (QTL) for FAW resistance in maize have been identified
including one with a very large phenotypic effect in bin 9.03 (Brooks et
al., 2007; Womack et al., 2018; 2020). Genetic mechanisms causing FAW
resistance in maize can be due to morphological features such as
structural barriers (waxes or tougher cell walls); antibiosis, or the
creation of metabolites toxic or off-putting to the insects; or hormones
that call insect predators (McMullen et al., 2009). A previous study by
Davis et al. (1998) found that resistance in young plants was linked to
thickness of the cuticle and the epidermal cell wall complex, which was
nearly twice as thick in resistant than susceptible plants and contained
more hemicellulose. In addition, the inner whorl tissue from the
resistant inbreds were tougher than in the susceptible, and resistant
hybrids completed the transition from juvenile to adult tissues (which
are less palatable to insects) sooner than susceptible hybrids. FAW
larvae feeding on maize plants in the whorl stage of growth feed
primarily on the yellow-green portion of whorl leaves. Davis et al.
(1999) compared the sizes of larvae fed the yellow-green leaves of
resistant or susceptible hybrids, compared to the outer (green) or
innermost (yellow-white) portions of the leaves. Larvae feeding on the
susceptible hybrid were larger than those feeding on the resistant, and
larvae feeding on the yellow-green leaves in resistant (and to a lesser
extent, susceptible) plants were smaller compared to those feeding on
other portions within the whorl. Thus, it is hypothesized that there are
antibiosis compounds localized there, especially in resistant plants,
that protect the growing point of the seedling plants (Williams et al.,
1998).
Both QTL mapping and association mapping may identify genes and genomic
regions associated with a trait of interest. A QTL mapping experiment
will likely identify very long genomic regions associated with the trait
and will only find the alleles that happen to be segregating in the two
parents of the mapping population. Conversely, a genome-wide association
study (GWAS) will identify far more regions associated with a trait,
often precisely enough to narrow down to one or a few genes influencing
the trait, but rarely identifies large effect genes for highly
quantitative traits, and many genes may be missed (Flint-Garcia et al.,
2005). A metabolic pathway analysis of the output of a GWAS study may
remedy this, and may identify more useful genes than the GWAS, and these
genes will be grouped into pathways of known function (Tang et al.,
2017; Thrash et al., 2022; 2021). The spread of FAW outside its native
range has increased the importance of identifying genes and regions of
the maize genome contributing resistance to FAW both to facilitate
breeding of non-GMO lines with FAW resistance, and to identify genes as
candidates for gene editing. Thus, this study was undertaken to dissect
the genetic basis and physiological mechanisms of resistance to FAW in
maize. The objectives of this study were to identify resistant maize
lines and SNPs associated with FAW resistance via GWAS; to use this
information to identify metabolic pathways associated with FAW
resistance via PAST; validate the presence of one or more of these
metabolites in resistant vs. susceptible plants; to integrate these
pathways into specific mechanisms used by maize plants as protection
from FAW; and to compare these results to previous genomic and
metabolomic studies of insect resistance in maize.
Materials and Methods